Notch1 inhibitors for inducing apoptosis

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of Notch1. The compositions comprise oligonucleotides, targeted to nucleic acid encoding Notch1. Methods of using these compounds for modulation of Notch1 expression and for diagnosis and treatment of disease associated with expression Notch1 are provided.

INTRODUCTION

[0001] This application is a continuation-in-part of U.S. application Ser. No. 10/160,497 filed on May 30, 2002.

FIELD OF THE INVENTION

[0002] The invention relates to prevention and treatment of diseases and conditions associated with insufficient apoptosis. This is accomplished through use of inhibitors of use of Notch1 inhibitors for inducing apoptosis is also provided.

BACKGROUND OF THE INVENTION

[0003] Intrinsic, cell-autonomous factors as well as non-autonomous, short-range and long-range signals guide cells through distinct developmental paths. An organism frequently uses the same signaling pathway within different cellular contexts to achieve unique developmental goals.

[0004] Notch signaling is an evolutionarily conserved mechanism used to control cell fates through local cell interactions. The gene encoding the original Notch receptor was discovered in Drosophila melanogaster due to the fact that partial loss of function of the gene results in notches at the wing margin (Artavanis-Tsakonas et al., Science, 1999, 284, 770-776). Signals transmitted through the Notch receptor, in combination with other cellular factors, influence differentiation, proliferation and apoptotic events at all stages of development (Artavanis-Tsakonas et al., Science, 1999, 284, 770-776).

[0005] Mature Notch proteins are heterodimeric receptors derived from the cleavage of Notch pre-proteins into an extracellular subunit containing multiple EGF-like repeats and a transmembrane subunit including the intracellular region (Blaumueller et al., Cell, 1997, go, 281-291). Notch activation results from the binding of ligands expressed by neighboring cells or soluble ligands and signaling from activated Notch involves networks of transcription regulators (Artavanis-Tsakonas et al., Science, 1995, 268, 225-232).

[0006] In context of experimental cancer immunotherapy, the Notch signaling network is acquiring increasing importance for its possible roles in neoplastic cells and the immune system (Jang et al., Curr. Opin. Mol. Ther., 2000, 2, 55-65).

[0007] Four mammalian Notch homologs have been identified and are designated Notch1, Notch2, Notch3 and Notch4. Human Notch1 (also known as Notch gene homolog 1 and TAN-1) was first identified in 1991 and later mapped to chromosome 9q34, a region associated with neoplasia-associated translocations (Ellisen et al., Cell, 1991, 66, 649-661; Larsson et al., Genomics, 1994, 24, 253-258). Larsson et al. predicted that the human Notch genes are proto-oncogenes and candidates for sites of chromosome breakage in neoplasia-associated translocations (Larsson et al., Genomics, 1994, 24, 253-258). Notch1 is expressed in many human tissues but is particularly abundant in lymphoid tissues (Ellisen et al., Cell, 1991, 66, 649-661).

[0008] An expressed sequence tag has been identified which represents a possible variant of Notch1, herein designated Notch1-B which starts in exon 27 and continues into intron 28.

[0009] Disclosed and claimed in U.S. Pat. No. 5,789,195 are nucleic acid sequences encoding Notch genes. Antibodies to human Notch proteins are additionally provided (Artavanis-Tsakonas et al., 1998). Amino acid sequences of Notch genes and antibodies against Notch proteins are also disclosed and claimed in U.S. Pat. No. 6,090,922 (Artavanis-Tsakonas et al., 2000).

[0010] Modulation of expression of Notch genes may prove to be a useful point for therapeutic intervention in developmental, hyperproliferative or autoimmune disorders or disorders arising from aberrant apoptosis.

[0011] Methods for producing allergen- or antigen-tolerant T-cells employing compositions capable of upregulating expression of an endogenous Notch protein are disclosed and claimed in PCT publication WO 00/36089 (Lamb et al., 2000).

[0012] Disclosed and claimed in U.S. Pat. No. 6,149,902 is a method for cell transplantation which includes contacting a precursor cell with an agonist of Notch1 function effective to inhibit differentiation of the cell wherein said agonist is a Delta protein, a Serrate protein or an antibody to a Notch protein (Artavanis-Tsakonas et al., 2000).

[0013] Disclosed in U.S. Pat. No. 6,083,904 and PCT publication WO 94/07474 are therapeutic and diagnostic methods and compositions based on Notch proteins and nucleic acids, wherein antisense methods are generally disclosed (Artavanis-Tsakonas, 2000; Artavanis-Tsakonas et al., 1994).

[0014] Disclosed and claimed in U.S. Pat. No. 5,786,158 are methods and compositions for the detection of malignancy or nervous system disorders based on the level of Notch proteins or nucleic acids (Artavanis-Tsakonas et al., 1998).

[0015] A Notch1 antisense transgenic mouse has been engineered and employed in investigations of regulation of NF-kappa-B activity by Notch1 (Cheng et al., J. Immunol., 2001, 167, 4458-4467).

[0016] Transfections of antisense Notch1 RNA have been carried out in 3T3-L1 cells and in K562 erythroleukemic cells in investigations of the roles of Notch1 in adipogenesis, ras signaling and megakaryocytic differentiation (Garces et al., J. Biol. Chem., 1997, 272, 29729-29734; Lam et al., J. Biol. Chem., 2000, 275, 19676-19684; Ruiz-Hidalgo et al., Int. J. Oncol., 1999, 14, 777-783).

[0017] A Notch1 antisense oligonucleotide has been employed in a study of the role of Notch1 in tumor necrosis factor-alpha-induced proliferation of human synoviocytes (Nakazawa et al., Arthritis Rheum., 2001, 44, 1545-1554).

[0018] Austin et al. have investigated the role of Notch1 in development of retinal ganglion cells by employing Notch1 antisense phosphorothioate oligonucleotides designed against three distinct regions of the chicken Notch1 sequence: the EGF repeat region, the lin12/notch region and the cdc10/ankyrin repeat region (Austin et al., Development, 1995, 121, 3637-3650). These same oligonucleotides have subsequently been employed in further investigations of the role of Notch1 in retinal cell development and in auditory hair cell and neuronal differentiation (Faux et al., J. Neurosci., 2001, 21, 5587-5596; Waid and McLoon, Development, 1998, 125, 1059-1066; Zine et al., Development, 2000, 127, 3373-3383.).

[0019] Disclosed and claimed in PCT publication WO 01/25422 and Japanese Patent JP13122787 are antisense oligonucleotides directed to translational start codon and exon 28 of human Notch1, respectively (Bartelmez and Iversen, 2001; Nakajima and Nishioka, 2001).

[0020] Disclosed and claimed in PCT publication WO 00/20576 are methods for inducing differentiation and apoptosis in human cells that over express Notch proteins wherein Notch function is disrupted using antisense oligonucleotides that target the EGF repeat region, the lin/notch region and the ankyrin region (Miele et al., 2000). These same oligonucleotides have also been employed in an investigation of the role of Notch1 in murine erythroleukemia cell apoptosis (Shelly et al., J. Cell. Biochem., 1999, 73, 164-175).

[0021] Currently, there are no known therapeutic agents that effectively inhibit the synthesis of Notch1. To date, investigative strategies aimed at modulating Notch1 expression have involved the use of antibodies and Notch-regulating proteins as well as antisense RNA and oligonucleotides. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting Notch1 function.

[0022] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of expression of Notch1.

[0023] The present invention provides compositions and methods for modulating expression of Notch1, including expression of variants of Notch1.

SUMMARY OF THE INVENTION

[0024] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding Notch1, and which modulate the expression of Notch1. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of Notch1 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of Notch1 by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding Notch1, ultimately modulating the amount of Notch1 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Notch1. As used herein, the terms “target nucleic acid” and “nucleic acid encoding Notch1” encompass DNA encoding Notch1, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of Notch1. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.

[0026] It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding Notch1. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding Notch1, regardless of the sequence(s) of such codons.

[0027] It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon.

[0028] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.

[0029] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It has also been found that introns can be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

[0030] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and extronic regions.

[0031] Upon excision of one or more exon or intron regions or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0032] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites.

[0033] Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0034] In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.

[0035] An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. It is preferred that the antisense compounds of the present invention comprise at least 80% sequence complementarity to a target region within the target nucleic acid, moreover that they comprise 90% sequence complementarity and even more comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al, J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0036] Antisense and other compounds of the invention, which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The sites to which these preferred antisense compounds are specifically hybridizable are hereinbelow referred to as “preferred target regions” and are therefore preferred sites for targeting. As used herein the term “preferred target region” is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target regions represent regions of the target nucleic acid which are accessible for hybridization.

[0037] While the specific sequences of particular preferred target regions are set forth below, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target regions may be identified by one having ordinary skill.

[0038] Target regions 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target regions are considered to be suitable preferred target regions as well.

[0039] Exemplary good preferred target regions include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred target regions (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly good preferred target regions are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred target regions (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art, once armed with the empirically-derived preferred target regions illustrated herein will be able, without undue experimentation, to identify further preferred target regions. In addition, one having ordinary skill in the art will also be able to identify additional compounds, including oligonucleotide probes and primers, that specifically hybridize to these preferred target regions using techniques available to the ordinary practitioner in the art.

[0040] Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.

[0041] For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in combination with other antisense compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0042] Expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0043] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (reviewed in To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0044] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.

[0045] In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

[0046] While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides from about 8 to about 50 nucleobases, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.

[0047] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0048] Exemplary preferred antisense compounds include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art, once armed with the empirically-derived preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.

[0049] Antisense and other compounds of the invention, which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are herein identified as preferred embodiments of the invention. While specific sequences of the antisense compounds are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred antisense compounds may be identified by one having ordinary skill.

[0050] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. In addition, linear structures may also have internal nucleobase complementarity and may therefore fold in a manner as to produce a double stranded structure. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0051] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0052] Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0053] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0054] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0055] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0056] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0057] Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃) —CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0058] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N— alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylamino-ethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0059] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0060] A further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0061] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyl-adenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0062] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0063] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include inter-calators, reporter molecules, polyamines, polyamides, poly-ethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmaco-dynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0064] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0065] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as interferon-induced RNAseL which cleaves both cellular and viral RNA. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0066] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0067] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0068] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0069] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivaients.

[0070] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0071] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

[0072] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci., 1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.

[0073] For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

[0074] The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of Notch1 is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.

[0075] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding Notch1, enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding Notch1 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of Notch1 in a sample may also be prepared.

[0076] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

[0077] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Preferred topical formulations include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.

[0078] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide completing agents include

[0079] poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. applications Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.

[0080] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0081] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0082] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0083] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0084] In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

[0085] Emulsions

[0086] The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

[0087] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0088] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

[0089] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

[0090] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0091] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

[0092] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0093] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

[0094] In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0095] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

[0096] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C₈-C₁₂) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C₈-C₁₀ glycerides, vegetable oils and silicone oil.

[0097] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

[0098] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

[0099] Liposomes

[0100] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

[0101] Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

[0102] In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

[0103] Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0104] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

[0105] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

[0106] Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.

[0107] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0108] Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0109] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidyicholine and/or cholesterol.

[0110] Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0111] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

[0112] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765). Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).

[0113] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C₁₂15G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

[0114] A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. Wo 97/04787 to Love et al. discloses liposomes comprising antisense oligonucleotides targeted to the raf gene.

[0115] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0116] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0117] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0118] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0119] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[0120] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0121] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285)

[0122] Penetration Enhancers

[0123] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

[0124] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

[0125] Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0126] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0127] Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0128] Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0129] Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0130] Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.

[0131] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

[0132] Carriers

[0133] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0134] Excipients

[0135] In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0136] Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0137] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

[0138] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0139] Other Components

[0140] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0141] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0142] Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0143] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0144] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0145] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1

[0146] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy Amidites

[0147] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2′-alkoxy amidites, optimized synthesis cycles were developed that incorporate multiple steps coupling longer wait times relative to standard synthesis cycles.

[0148] The following abbreviations are used in the text: thin layer chromatography (TLC), melting point (MP), high pressure liquid chromatography (HPLC), Nuclear Magnetic Resonance (NMR), argon (Ar), methanol (MeOH), dichloromethane (CH₂Cl₂), triethylamine (TEA), dimethyl formamide (DMF), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF).

[0149] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-dC) nucleotides were synthesized according to published methods (Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.) or prepared as follows:

[0150] Preparation of 5′-O-Dimethoxytrityl-thymidine Intermediate for 5-methyl dC Amidite

[0151] To a 50 L glass reactor equipped with air stirrer and Ar gas line was added thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 L) at ambient temperature. Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol, 1.05 eq) was added as a solid in four portions over 1 h. After 30 min, TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent and by-products and 2% 3′,5′-bis DMT product (R_(f) in EtOAc 0.45, 0.05, 0.98, 0.95 respectively). Saturated sodium bicarbonate (4 L) and CH₂Cl₂ were added with stirring (pH of the aqueous layer 7.5). An additional 18 L of water was added, the mixture was stirred, the phases were separated, and the organic layer was transferred to a second 50 L vessel. The aqueous layer was extracted with additional CH₂Cl₂ (2×2 L). The combined organic layer was washed with water (10 L) and then concentrated in a rotary evaporator to approx. 3.6 kg total weight. This was redissolved in CH₂Cl₂ (3.5 L), added to the reactor followed by water (6 L) and hexanes (13 L). The mixture was vigorously stirred and seeded to give a fine white suspended solid starting at the interface. After stirring for 1 h, the suspension was removed by suction through a ½″ diameter teflon tube into a 20 L suction flask, poured onto a 25 cm Coors Buchner funnel, washed with water (2×3 L) and a mixture of hexanes-CH₂Cl₂ (4:1, 2×3 L) and allowed to air dry overnight in pans (1″ deep). This was further dried in a vacuum oven (75° C., 0.1 mm Hg, 48 h) to a constant weight of 2072 g (93%) of a white solid, (mp 122-124° C.). TLC indicated a trace contamination of the bis DMT product. NMR spectroscopy also indicated that 1-2 mole percent pyridine and about 5 mole percent of hexanes was still present.

[0152] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine Intermediate for 5-methyl-dC Amidite

[0153] To a 50 L Schott glass-lined steel reactor equipped with an electric stirrer, reagent addition pump (connected to an addition funnel), heating/cooling system, internal thermometer and an Ar gas line was added 5′-O-dimethoxytrityl-thymidine (3.00 kg, 5.51 mol), anhydrous acetonitrile (25 L) and TEA (12.3 L, 88.4 mol, 16 eq). The mixture was chilled with stirring to −10° C. internal temperature (external −20° C.). Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30 minutes while maintaining the internal temperature below −5° C., followed by a wash of anhydrous acetonitrile (1 L). Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition. The reaction was allowed to warm to 0° C. and the reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; R_(f) 0.43 to 0.84 of starting material and silyl product, respectively). Upon completion, triazole (3.05 kg, 44 mol, 8.0 eq) was added the reaction was cooled to −20° C. internal temperature (external −30° C.). Phosphorous oxychloride (1035 mL, 11.1 mol, 2.01 eq) was added over 60 min so as to maintain the temperature between −20° C. and −10° C. during the strongly exothermic process, followed by a wash of anhydrous acetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1 h. TLC indicated a complete conversion to the triazole product (R_(f) 0.83 to 0.34 with the product spot glowing in long wavelength UV light). The reaction mixture was a peach-colored thick suspension, which turned darker red upon warming without apparent decomposition. The reaction was cooled to −15° C. internal temperature and water (5 L) was slowly added at a rate to maintain the temperature below +10° C. in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic). Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2×8 L). The combined water layers were back-extracted with EtOAc (6 L). The water layer was discarded and the organic layers were concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam). The second half of the reaction was treated in the same way. Each residue was dissolved in dioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight (although the reaction is complete within 1 h).

[0154] TLC indicated a complete reaction (product R_(f) 0.35 in EtOAc-MeOH 4:1). The reaction solution was concentrated on a rotary evaporator to a dense foam. Each foam was slowly redissolved in warm EtOAc (4 L; 50° C.), combined in a 50 L glass reactor vessel, and extracted with water (2×4L) to remove the triazole by-product. The water was back-extracted with EtOAc (2 L). The organic layers were combined and concentrated to about 8 kg total weight, cooled to 0° C. and seeded with crystalline product. After 24 hours, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc (3×3L) until a white powder was left and then washed with ethyl ether (2×3L). The solid was put in pans (1″ deep) and allowed to air dry overnight. The filtrate was concentrated to an oil, then redissolved in EtOAc (2 L), cooled and seeded as before. The second crop was collected and washed as before (with proportional solvents) and the filtrate was first extracted with water (2×1L) and then concentrated to an oil. The residue was dissolved in EtOAc (1 L) and yielded a third crop which was treated as above except that more washing was required to remove a yellow oily layer.

[0155] After air-drying, the three crops were dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g, respectively) and combined to afford 2550 g (85%) of a white crystalline product (MP 215-217° C.) when TLC and NMR spectroscopy indicated purity. The mother liquor still contained mostly product (as determined by TLC) and a small amount of triazole (as determined by NMR spectroscopy), bis DMT product and unidentified minor impurities. Tf desired, the mother liquor can be purified by silica gel chromatography using a gradient of MeOH (0-25%) in EtOAc to further increase the yield.

[0156] Preparation of 51-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine Penultimate Intermediate for 5-methyl dC Amidite

[0157] Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000 g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambient temperature in a 50 L glass reactor vessel equipped with an air stirrer and argon line. Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86 mol, 1.05 eq) was added and the reaction was stirred at ambient temperature for 8 h. TLC (CH₂Cl₂-EtOAc; CH₂Cl₂-EtOAc 4:1; R_(f) 0.25) indicated approx. 92% complete reaction. An additional amount of benzoic anhydride (44 g, 0.19 mol) was added. After a total of 18 h, TLC indicated approx. 96% reaction completion. The solution was diluted with EtOAc (20 L), TEA (1020 mL, 7.36 mol, ca 2.0 eq) was added with stirring, and the mixture was extracted with water (15 L, then 2×10 L). The aqueous layer was removed (no back-extraction was needed) and the organic layer was concentrated in 2×20 L rotary evaporator flasks until a foam began to form. The residues were coevaporated with acetonitrile (1.5 L each) and dried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a dense foam. High pressure liquid chromatography (HPLC) revealed a contamination of 6.3% of N4, 3′-O-dibenzoyl product, but very little other impurities.

[0158] THe product was purified by Biotage column chromatography (5 kg Biotage) prepared with 65:35:1 hexanes-EtOAc-TEA (4L). The crude product (800 g),dissolved in CH₂Cl₂ (2 L), was applied to the column. The column was washed with the 65:35:1 solvent mixture (20 kg), then 20:80:1 solvent mixture (10 kg), then 99:1 EtOAc:TEA (17 kg). The fractions containing the product were collected, and any fractions containing the product and impurities were retained to be resubjected to column chromatography. The column was re-equilibrated with the original 65:35:1 solvent mixture (17 kg). A second batch of crude product (840 g) was applied to the column as before. The column was washed with the following solvent gradients: 65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1 (10 kg), and 99:1 EtOAc:TEA(15 kg). The column was reequilibrated as above, and a third batch of the crude product (850 g) plus impure fractions recycled from the two previous columns (28 g) was purified following the procedure for the second batch. The fractions containing pure product combined and concentrated on a 20L rotary evaporator, co-evaporated with acetontirile (3 L) and dried (0.1 mm Hg, 48 h, 25° C.) to a constant weight of 2023 g (85%) of white foam and 20 g of slightly contaminated product from the third run. HPLC indicated a purity of 99.8% with the balance as the diBenzoyl product.

[0159] [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC Amidite)

[0160] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N-benzoyl-5-methylcytidine (998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (300 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (15 ml) was added and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2.5 L) and water (600 ml), and extracted with hexane (3×3 L). The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (7.5 L) and hexane (6 L). The two layers were separated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L) and water (3×2 L), and the phases were separated. The organic layer was dried (Na₂SO₄), filtered and rotary evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried to a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 g an off-white foam solid (96%).

[0161] 2′-Fluoro Amidites

[0162] 2′-Fluorodeoxyadenosine Amidites

[0163] 2′-fluoro oligonucleotides were synthesized as described previously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No. 5,670,633, herein incorporated by reference. The preparation of 2′-fluoropyrimidines containing a 5-methyl substitution are described in U.S. Pat. No. 5,861,493. Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and whereby the 2′-alpha-fluoro atom is introduced by a SN2-displacement of a 2′-beta-triflate group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0164] 2′-Fluorodeoxyguanosine

[0165] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate isobutyryl-arabinofuranosylguanosine. Alternatively, isobutyryl-arabinofuranosylguanosine was prepared as described by Ross et al., (Nucleosides & Nucleosides, 16, 1645, 1997). Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give isobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.

[0166] 2′-Fluorouridine

[0167] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a literature procedure in which 2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0168] 2′-Fluorodeoxycytidine

[0169] 2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0170] 2′-O-(2-Methoxyethyl) Modified Amidites

[0171] 2′-O-Methoxyethyl-substituted nucleoside amidites (otherwise known as MOE amidites) are prepared as follows, or alternatively, as per the methods of Martin, P., (Helvetica Chimica Acta, 1995, 78, 486-504).

[0172] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate

[0173] 2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol), tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60 g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12 L three necked flask and heated to 130° C. (internal temp) at atmospheric pressure, under an argon atmosphere with stirring for 21 h. TLC indicated a complete reaction. The solvent was removed under reduced pressure until a sticky gum formed (50-85° C. bath temp and 100-11 mm Hg) and the residue was redissolved in water (3 L) and heated to boiling for 30 min in order the hydrolyze the borate esters. The water was removed under reduced pressure until a foam began to form and then the process was repeated. HPLC indicated about 77% product, 15% dimer (5′ of product attached to 2′ of starting material) and unknown derivatives, and the balance was a single unresolved early eluting peak.

[0174] The gum was redissolved in brine (3 L), and the flask was rinsed with additional brine (3 L). The combined aqueous solutions were extracted with chloroform (20 L) in a heavier-than continuous extractor for 70 h. The chloroform layer was concentrated by rotary evaporation in a 20 L flask to a sticky foam (2400 g). This was coevaporated with MeOH (400 mL) and EtOAc (8 L) at 75° C. and 0.65 atm until the foam dissolved at which point the vacuum was lowered to about 0.5 atm. After 2.5 L of distillate was collected a precipitate began to form and the flask was removed from the rotary evaporator and stirred until the suspension reached ambient temperature. EtOAc (2 L) was added and the slurry was filtered on a 25 cm table top Buchner funnel and the product was washed with EtOAc (3×2 L). The bright white solid was air dried in pans for 24 h then further dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) to afford 1649 g of a white crystalline solid (mp 115.5-116.5° C.).

[0175] The brine layer in the 20 L continuous extractor was further extracted for 72 h with recycled chloroform. The chloroform was concentrated to 120 g of oil and this was combined with the mother liquor from the above filtration (225 g), dissolved in brine (250 mL) and extracted once with chloroform (250 mL). The brine solution was continuously extracted and the product was crystallized as described above to afford an additional 178 g of crystalline product containing about 2% of thymine. The combined yield was 1827 g (69.4%). HPLC indicated about 99.5% purity with the balance being the dimer.

[0176] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine Penultimate Intermediate

[0177] In a 50 L glass-lined steel reactor, 2′-O-(2-methoxyethyl)-5-methyl-uridine (MOE-T, 1500 g, 4.738 mol), lutidine (1015 g, 9.476 mol) were dissolved in anhydrous acetonitrile (15 L). The solution was stirred rapidly and chilled to −10° C. (internal temperature). Dimethoxytriphenylmethyl chloride (1765.7 g, 5.21 mol) was added as a solid in one portion. The reaction was allowed to warm to −2° C. over 1 h. (Note: The reaction was monitored closely by TLC (EtOAc) to determine when to stop the reaction so as to not generate the undesired bis-DMT substituted side product). The reaction was allowed to warm from −2 to 3° C. over 25 min. then quenched by adding MeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L). The solution was transferred to a clear 50 L vessel with a bottom outlet, vigorously stirred for 1 minute, and the layers separated. The aqueous layer was removed and the organic layer was washed successively with 10% aqueous citric acid (8 L) and water (12 L). The product was then extracted into the aqueous phase by washing the toluene solution with aqueous sodium hydroxide (0.5N, 16 L and 8 L). The combined aqueous layer was overlayed with toluene (12 L) and solid citric acid (8 moles, 1270 g) was added with vigorous stirring to lower the pH of the aqueous layer to 5.5 and extract the product into the toluene. The organic layer was washed with water (10 L) and TLC of the organic layer indicated a trace of DMT-O-Me, bis DMT and dimer DMT.

[0178] The toluene solution was applied to a silica gel column (6 L sintered glass funnel containing approx. 2 kg of silica gel slurried with toluene (2 L) and TEA(25 mL)) and the fractions were eluted with toluene (12 L) and EtOAc (3×4 L) using vacuum applied to a filter flask placed below the column. The first EtOAc fraction containing both the desired product and impurities were resubjected to column chromatography as above. The clean fractions were combined, rotary evaporated to a foam, coevaporated with acetonitrile (6 L) and dried in a vacuum oven (0.1 mm Hg, 40 h, 40° C.) to afford 2850 g of a white crisp foam. NMR spectroscopy indicated a 0.25 mole % remainder of acetonitrile (calculates to be approx. 47 g) to give a true dry weight of 2803 g (96%). HPLC indicated that the product was 99.41% pure, with the remainder being 0.06 DMT-O-Me, 0.10 unknown, 0.44 bis DMT, and no detectable dimer DMT or 3′-O-DMT.

[0179] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T Amidite)

[0180] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridine (1237 g, 2.0 mol) was dissolved in anhydrous DMF (2.5 L). The solution was co-evaporated with toluene (200 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g, 1.0 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (20 ml) was added and the solution was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (3.5 L) and water (600 ml) and extracted with hexane (3×3L). The mixture was diluted with water (1.6 L) and extracted with the mixture of toluene (12 L) and hexanes (9 L). The upper layer was washed with DMF-water (7:3 v/v, 3×3 L) and water (3×3 L). The organic layer was dried (Na₂SO₄), filtered and evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1526 g of an off-white foamy solid (95%).

[0181] Preparation of 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate

[0182] To a 50 L Schott glass-lined steel reactor equipped with an electric stirrer, reagent addition pump (connected to an addition funnel), heating/cooling system, internal thermometer and argon gas line was added 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-uridine (2.616 kg, 4.23 mol, purified by base extraction only and no scrub column), anhydrous acetonitrile (20 L), and TEA (9.5 L, 67.7 mol, 16 eq). The mixture was chilled with stirring to −10° C. internal temperature (external −20° C.). Trimethylsilylchloride (1.60 L, 12.7 mol, 3.0 eq) was added over 30 min. while maintaining the internal temperature below −5° C., followed by a wash of anhydrous acetonitrile (1 L). (Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition). The reaction was allowed to warm to 0° C. and the reaction progress was confirmed by TLC (EtOAc, R_(f) 0.68 and 0.87 for starting material and silyl product, respectively). Upon completion, triazole (2.34 kg, 33.8 mol, 8.0 eq) was added the reaction was cooled to −20° C. internal temperature (external −30° C.). Phosphorous oxychloride (793 mL, 8.51 mol, 2.01 eq) was added slowly over 60 min so as to maintain the temperature between −20° C. and −10° C. (note: strongly exothermic), followed by a wash of anhydrous acetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1 h, at which point it was an off-white thick suspension. TLC indicated a complete conversion to the triazole product (EtOAc, R_(f) 0.87 to 0.75 with the product spot glowing in long wavelength UV light). The reaction was cooled to −15° C. and water (5 L) was slowly added at a rate to maintain the temperature below +10° C. in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic). Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2×8 L). The second half of the reaction was treated in the same way. The combined aqueous layers were back-extracted with EtOAc (8 L) The organic layers were combined and concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam). The residue was dissolved in dioxane (2 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight

[0183] TLC indicated a complete reaction (CH₂Cl₂-acetone-MeOH, 20:5:3, R_(f) 0.51). The reaction solution was concentrated on a rotary evaporator to a dense foam and slowly redissolved in warm CH₂Cl₂ (4 L, 40° C.) and transferred to a 20 L glass extraction vessel equipped with a air-powered stirrer. The organic layer was extracted with water (2×6 L) to remove the triazole by-product. (Note: In the first extraction an emulsion formed which took about 2 h to resolve). The water layer was back-extracted with CH₂Cl₂ (2×2 L), which in turn was washed with water (3 L). The combined organic layer was concentrated in 2×20 L flasks to a gum and then recrystallized from EtOAc seeded with crystalline product. After sitting overnight, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc until a white free-flowing powder was left (about 3×3 L). The filtrate was concentrated to an oil recrystallized from EtOAc, and collected as above. The solid was air-dried in pans for 48 h, then further dried in a vacuum oven (50° C., 0.1 mm Hg, 17 h) to afford 2248 g of a bright white, dense solid (86%). An HPLC analysis indicated both crops to be 99.4% pure and NMR spectroscopy indicated only a faint trace of EtOAc remained.

[0184] Preparation of 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidine Penultimate Intermediate:

[0185] Crystalline 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-cytidine (1000 g, 1.62 mol) was suspended in anhydrous DMF (3 kg) at ambient temperature and stirred under an Ar atmosphere. Benzoic anhydride (439.3 g, 1.94 mol) was added in one portion. The solution clarified after 5 hours and was stirred for 16 h. HPLC indicated 0.45% starting material remained (as well as 0.32% N4, 3′-O-bis Benzoyl). An additional amount of benzoic anhydride (6.0 g, 0.0265 mol) was added and after 17 h, HPLC indicated no starting material was present. TEA (450 mL, 3.24 mol) and toluene (6 L) were added with stirring for 1 minute. The solution was washed with water (4×4 L), and brine (2×4 L). The organic layer was partially evaporated on a 20 L rotary evaporator to remove 4 L of toluene and traces of water. HPLC indicated that the bis benzoyl side product was present as a 6% impurity. The residue was diluted with toluene (7 L) and anhydrous DMSO (200 mL, 2.82 mol) and sodium hydride (60% in oil, 70 g, 1.75 mol) was added in one portion with stirring at ambient temperature over 1 h. The reaction was quenched by slowly adding then washing with aqueous citric acid (10%, 100 mL over 10 min, then 2×4 L), followed by aqueous sodium bicarbonate (?%, 2 L), water (2×4 L) and brine (4 L). The organic layer was concentrated on a 20 L rotary evaporator to about 2 L total volume. The residue was purified by silica gel column chromatography (6 L Buchner funnel containing 1.5 kg of silica gel wetted with a solution of EtOAc-hexanes-TEA(70:29:1)). The product was eluted with the same solvent (30 L) followed by straight EtOAc (6 L). The fractions containing the product were combined, concentrated on a rotary evaporator to a foam and then dried in a vacuum oven (50° C., 0.2 mm Hg, 8 h) to afford 1155 g of a crisp, white foam (98%). HPLC indicated a purity of >99.7%.

[0186] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C Amidite)

[0187] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidine (1082 g, 1.5 mol) was dissolved in anhydrous DMF (2 L) and co-evaporated with toluene (300 ml) at 50° C. under reduced pressure. The mixture was cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (1 L) and water (400 ml) and extracted with hexane (3×3 L). The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (9 L) and hexanes (6 L). The two layers were separated and the upper layer was washed with DMF-water (60:40 v/v, 3×3 L) and water (3×2 L). The organic layer was dried (Na₂SO₄), filtered and evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1336 g of an off-white foam (97%).

[0188] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A Amdite)

[0189] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosine (purchased from Reliable Biopharmaceutical, St. Lois, Mo.), 1098 g, 1.5 mol) was dissolved in anhydrous DMF (3 L) and co-evaporated with toluene (300 ml) at 50° C. The mixture was cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (78.8 g, 1.24 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (1 L) and water (400 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (1.4 L) and extracted with the mixture of toluene (9 L) and hexanes (6 L). The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L) and water (3×2 L). The organic layer was dried (Na₂SO₄), filtered and evaporated to a sticky foam. The residue was co-evaporated with acetonitrile (2.5 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1350 g of an off-white foam solid (96%).

[0190] Prepartion of [5′-O— (4,4-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G Amidite)

[0191] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrlguanosine (purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (200 ml) at 50° C., cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2 L) and water (600 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (2 L) and extracted with a mixture of toluene (10 L) and hexanes (5 L). The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and the solution was washed with water (3×4 L). The organic layer was dried (Na₂SO₄), filtered and evaporated to approx. 4 kg. Hexane (4 L) was added, the mixture was shaken for 10 min, and the supernatant liquid was decanted. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1660 g of an off-white foamy solid (91%).

[0192] 2′-O-(Aminooxyethyl) Nucleoside Amidites and 2′-O-(dimethylaminooxyethyl) Nucleoside Amidites

[0193] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

[0194] 2′-(Dimethylaminooxyethoxy) nucleoside amidites (also known in the art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine.

[0195] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0196] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (R_(f) 0.22, EtOAc) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between CH₂Cl₂ (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of EtOAc and ethyl ether (600 mL) and cooling the solution to −10° C. afforded a white crystalline solid which was collected by filtration, washed with ethyl ether (3×2 00 mL) and dried (40° C., 1 mm Hg, 24 h) to afford 149 g of white solid (74.8%). TLC and NMR spectroscopy were consistent with pure product.

[0197] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0198] In the fume hood, ethylene glycol (350 mL, excess) was added cautiously with manual stirring to a 2 L stainless steel pressure reactor containing borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). (Caution: evolves hydrogen gas). 5′-O-tert-Butyldiphenylsilyl-O2-2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160° C. was reached and then maintained for 16 h (pressure <100 psig). The reaction vessel was cooled to ambient temperature and opened. TLC (EtOAc, R_(f) 0.67 for desired product and R_(f) 0.82 for ara-T side product) indicated about 70% conversion to the product. The solution was concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol. (Alternatively, once the THF has evaporated the solution can be diluted with water and the product extracted into EtOAc). The residue was purified by column chromatography (2 kg silica gel, EtOAc-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, evaporated and dried to afford 84 g of a white crisp foam (50%), contaminated starting material (17.4 g, 12% recovery) and pure reusable starting material (20 g, 13% recovery). TLC and NMR spectroscopy were consistent with 99% pure product.

[0199] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0200] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol) and dried over P₂O₅ under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle). Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture with the rate of addition maintained such that the resulting deep red coloration is just discharged before adding the next drop. The reaction mixture was stirred for 4 hrs., after which time TLC (EtOAc:hexane, 60:40) indicated that the reaction was complete. The solvent was evaporated in vacuuo and the residue purified by flash column chromatography (eluted with 60:40 EtOAc:hexane), to yield 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%) upon rotary evaporation.

[0201] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0202] 2′-O-([2-phthalimidoxy)ethyl]-5′-L-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h the mixture was filtered, the filtrate washed with ice cold CH₂Cl₂, and the combined organic phase was washed with water and brine and dried (anhydrous Na₂SO₄). The solution was filtered and evaporated to afford 2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). Formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was stirred for 1 h. The solvent was removed under vacuum and the residue was purified by column chromatography to yield 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%) upon rotary evaporation. 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine

[0203] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL) and cooled to 10° C. under inert atmosphere. Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and the reaction mixture was stirred. After 10 minutes the reaction was warmed to room temperature and stirred for 2 h. while the progress of the reaction was monitored by TLC (5% MeOH in CH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and the product was extracted with EtOAc (2×20 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. This entire procedure was repeated with the resulting residue, with the exception that formaldehyde (20% w/w, 30 mL, 3.37 mol) was added upon dissolution of the residue in the PPTS/MeOH solution. After the extraction and evaporation, the residue was purified by flash column chromatography and (eluted with 5% MeOH in CH₂Cl₂) to afford 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%) upon rotary evaporation.

[0204] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0205] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and TEA (1.67 mL, 12 mmol, dry, stored over KOH) and added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol). The reaction was stirred at room temperature for 24 hrs and monitored by TLC (5% MeOH in CH₂Cl₂). The solvent was removed under vacuum and the residue purified by flash column chromatography (eluted with 10% MeOH in CH₂Cl₂) to afford 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%) upon rotary evaporation of the solvent.

[0206] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0207] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P₂O₅ under high vacuum overnight at 40° C., co-evaporated with anhydrous pyridine (20 mL), and dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol) and 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) were added to the pyridine solution and the reaction mixture was stirred at room temperature until all of the starting material had reacted. Pyridine was removed under vacuum and the residue was purified by column chromatography (eluted with 10% MeOH in CH₂Cl₂ containing a few drops of pyridine) to yield 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%) upon rotary evaporation.

[0208] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0209] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried over P₂O₅ under high vacuum overnight at 40° C. This was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 h under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, then the residue was dissolved in EtOAc (70 mL) and washed with 5% aqueous NaHCO₃ (40 mL). The EtOAc layer was dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue obtained was purified by column chromatography (EtOAc as eluent) to afford 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%) upon rotary evaporation.

[0210] 2′-(Aminooxyethoxy) Nucleoside Amidites

[0211] 2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as 2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.

[0212] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramiditel

[0213] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may be phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0214] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

[0215] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, or 2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside amidites are prepared similarly.

[0216] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine

[0217] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) was slowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. (Caution: Hydrogen gas evolves as the solid dissolves). O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) were added and the bomb was sealed, placed in an oil bath and heated to 155° C. for 26 h. then cooled to room temperature. The crude solution was concentrated, the residue was diluted with water (200 mL) and extracted with hexanes (200 mL). The product was extracted from the aqueous layer with EtOAc (3×200 mL) and the combined organic layers were washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (eluted with 5:100:2 MeOH/CH₂Cl₂/TEA) as the eluent. The appropriate fractions were combined and evaporated to afford the product as a white solid.

[0218] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyl Uridine

[0219] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. The reaction mixture was poured into water (200 mL) and extracted with CH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers were washed with saturated NaHCO₃ solution, followed by saturated NaCl solution, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography (eluted with 5:100:1 MeOH/CH₂Cl₂/TEA) to afford the product.

[0220] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0221] Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) were added to a solution of 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere of argon. The reaction mixture was stirred overnight and the solvent evaporated. The resulting residue was purified by silica gel column chromatography with EtOAc as the eluent to afford the title compound.

Example 2

[0222] Oligonucleotide Synthesis

[0223] Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0224] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄oAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0225] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0226] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0227] Phosphoramidite oligonucleotides are prepared as described in U.S. Patent, 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0228] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0229] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0230] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0231] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3

[0232] Oligonucleoside Synthesis

[0233] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedi-methylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0234] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0235] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0236] PNA Synthesis

[0237] Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.

Example 5

[0238] Synthesis of Chimeric Oligonucleotides

[0239] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[0240] [2′-O—Me]—[2′-deoxy]—[2′-O—Me] Chimeric Phosphorothioate Oligonucleotides

[0241] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligo-nucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[0242] [2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O--(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0243] [2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[0244] [2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxy Phosphorothioate]—[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides

[0245] [2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxy phosphorothioatel—[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0246] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0247] Oligonucleotide Isolation

[0248] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32+/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

[0249] Oligonucleotide Synthesis—96 Well Plate Format

[0250] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0251] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

[0252] Oligonucleotide Analysis—96-Well Plate Format

[0253] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

[0254] Cell Culture and Oligonucleotide Treatment

[0255] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.

[0256] T-24 Cells:

[0257] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0258] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0259] A549 Cells:

[0260] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0261] NHDF Cells:

[0262] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0263] HEK Cells:

[0264] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0265] Treatment with Antisense Compounds:

[0266] When cells reached 70% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligbnucleotide. After 4-7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0267] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments.

Example 10

[0268] Analysis of Oligonucleotide Inhibition of Notch1 Expression

[0269] Antisense modulation of Notch1 expression can be assayed in a variety of ways known in the art. For example, Notch1 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISMT™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0270] Protein levels of Notch1 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to Notch1 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997). Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997).

[0271] Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998). Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997). Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991).

Example 11

[0272] Poly(A)+mRNA Isolation

[0273] Poly(A)+mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+mRNA isolation are taught in, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993). Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5° NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90 C hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0274] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Example 12

[0275] Total RNA Isolation

[0276] Total RNA was isolated using an RNEASY 96TM kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 170 μL water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0277] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

[0278] Real-Time Quantitative PCR Analysis of Notch1 mRNA Levels

[0279] Quantitation of Notch1 mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0280] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0281] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer (—MgCl2), 6.6 mM MgCl2, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5× ROX dye) to 96-well plates containing 30 μL total RNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0282] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). CAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0283] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.

[0284] Probes and primers to human Notch1 were designed to hybridize to a human Notch1 sequence, using published sequence information (the complement of residues 322000-377000 of GenBank accession number NT_(—)024000.7, representing a genomic sequence of Notch1, incorporated herein as SEQ ID NO:4). For human Notch1 the PCR primers were:

[0285] forward primer: CGGGTCCACCAGTTTGAAT (SEQ ID NO: 5)

[0286] reverse primer: TTGTATTGGTTCGGCACCAT (SEQ ID NO: 6) and the PCR probe was: FAM-TCCCGGCTGCAGAGCGG-TAMRA

[0287] (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were:

[0288] forward primer: CAAGGTGAAGGTCGGAGTC(SEQ ID NO:8)

[0289] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14

[0290] Northern Blot Analysis of Notch1 mRNA Levels

[0291] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0292] To detect human Notch1, a human Notch1 specific probe was prepared by PCR using the forward primer CGGGTCCACCAGTTTGAAT (SEQ ID NO: 5) and the reverse primer TTGTATTGGTTCGGCACCAT (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0293] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

[0294] Antisense Inhibition of Human Notch1 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0295] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human Notch1 RNA, using published sequences (the complement of residues 322000-377000 of GenBank accession number NT_(—)024000.7, representing a genomic sequence of Notch1, incorporated herein as SEQ ID NO: 4; GenBank accession number AF308602.1, incorporated herein as SEQ ID NO: 11, GenBank accession number AI802214.1, the complement of which is incorporated herein as SEQ ID NO: 12, and GenBank accession number BC013208.1, incorporated herein as SEQ ID NO: 13). The oligonucleotides are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human Notch1 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which A549 cells were treated with the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human Notch1 mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET SEQ ID TARGET % SEQ CONTROL ISIS # REGION NO SITE SEQUENCE INHIB ID NO SEQ ID NO 226818 Intron 4 20230 cgtgcgtccctcttagggtc 38 14 1 226819 Intron: 4 29652 cacagcagacctgggcaggc 40 15 1 Exon Junction 226820 Intron 4 40111 cagccctcccctaatgagac 6 16 1 226821 Intron: 4 40500 cggccacgcactgtgcaggc 70 17 1 Exon Junction 226822 Intron: 4 45449 acggtctcacctgcgggcac 19 18 1 Exon Junction 226823 Exon: 4 45660 tcacttgaggcccacggagt 46 19 1 Intron Junction 226824 Exon: 4 47322 cactgcctacctggaagaca 36 20 1 Intron Junction 226825 Intron 4 49376 accacctgcgtcaccacatt 55 21 1 226826 Coding 11 394 cacgatttccctgaccagcc 13 22 1 226827 Coding 11 454 aagggcaggcactggccacc 66 23 1 226828 Coding 11 781 ttgcagttgtttcctggaca 66 24 1 226829 Coding 11 904 ttctggcaggcatttggcat 59 25 1 226830 Coding 11 1093 tggcacagcagacctgtgcg 30 26 1 226831 Coding 11 1098 tgaggtggcacagcagacct 52 27 1 226832 Coding 11 1222 tccacgtcctggctgcaggc 83 28 1 226833 Coding 11 1399 tccccaatctggtccaggca 77 29 1 226834 Coding 11 1404 ggaactccccaatctggtcc 18 30 1 226835 Coding 11 1963 ccatcgatcttgtccagaca 78 31 1 226836 Coding 11 2246 gttgttgttgatgtcacagt 67 32 1 226837 Coding 11 2251 cactcgttgttgttgatgtc 68 33 1 226838 Coding 11 2788 ggcaggcagtcgcagaaggc 64 34 1 226839 Coding 11 2794 aagccgggcaggcagtcgca 76 35 1 226840 Coding 11 2887 gtgtagctgtccacgcagtc 8 36 1 226841 Coding 11 2897 gcaggtgcacgtgtagctgt 22 37 1 226842 Coding 11 3165 gcacaaggttctggcagttg 47 38 1 226843 Coding 11 3298 gcagccacctcacaggacac 0 39 1 226844 Coding 11 3345 gccctccatgctggcacagg 93 40 1 226845 Coding 11 3350 acagagccctccatgctggc 64 41 1 226846 Coding 11 3613 gggcaggagcacttgtaggt 38 42 1 226847 Coding 11 3870 ggcactcgcagtggaagtca 65 43 1 226848 Coding 11 4008 cctcgaagcccgcagggcac 28 44 1 226849 Coding 11 4207 tcggatgtgggctcacaggt 64 45 1 226850 Coding 11 4274 gtagtccaggatgtggcaca 66 46 1 226851 Coding 11 4279 aagctgtagtccaggatgtg 55 47 1 226852 Coding 11 4435 ttgaagttgagggagcagtc 26 48 1 226853 Coding 11 4440 ggtcattgaagttgagggag 33 49 1 226854 Coding 11 4459 tgcgtgcagttcttccaggg 40 50 1 226855 Coding 11 4464 gagactgcgtgcagttcttc 40 51 1 226856 Coding 11 4507 tggctgtcacagtggccgtc 53 52 1 226857 Coding 11 4512 tgcactggctgtcacagtgg 71 53 1 226858 Coding 11 4600 tggtccttgcagtactggtc 70 54 1 226859 Coding 11 4605 tgaagtggtccttgcagtac 40 55 1 226860 Coding 11 4797 ccacgttggtgtgcagcacg 70 56 1 226861 Coding 11 4802 gaagaccacgttggtgtgca 49 57 1 226862 Coding 11 4807 cgcttgaagaccacgttggt 64 58 1 226863 Coding 11 4837 gggaagatcatctgctggcc 20 59 1 226864 Coding 11 5068 ctctggaagcactgcgagga 77 60 1 226865 Coding 11 5073 tggcactctggaagcactgc 75 61 1 226866 Coding 11 5260 ttgcgggacagcagcacccc 66 62 1 226867 Coding 11 5290 aaccagagctggccatgctg 60 63 1 226868 Coding 11 5295 cagggaaccagagctggcca 44 64 1 226869 Coding 11 5452 aacttcttggtctccaggtc 56 65 1 226870 Coding 11 5457 accggaacttcttggtctcc 58 66 1 226871 Coding 11 5554 gcagacatgcgcaggtcagc 63 67 1 226872 Coding 11 5559 ccatggcagacatgcgcagg 69 68 1 226873 Coding 11 5762 gcggtctgtctggttgtgca 38 69 1 226874 Coding 11 5848 ttggcatctgcgctggcctc 60 70 1 226875 Coding 11 6030 tgatgaggtcctccagcatg 50 71 1 226876 Coding 11 6035 tgagttgatgaggtcctcca 73 72 1 226877 Coding 11 6076 gcggacttgcccaggtcatc 55 73 1 226878 Coding 11 6136 ccgttcttcaggagcacaac 66 74 1 226879 Coding 11 6257 atccgtgatgtcccggttgg 75 75 1 226880 Coding 11 6418 tagccgttgggcgagcagag 73 76 1 226881 Coding 11 6523 ctccgtgccttgaggtcctt 88 77 1 226882 Coding 11 6528 tcttcctccgtgccttgagg 82 78 1 226883 Coding 11 6550 cagcccttgccatcctggga 84 79 1 226884 Coding 11 7129 gtctgcaccaggtgaggctg 62 80 1 226885 Coding 11 7135 tgctgggtctgcaccaggtg 53 81 1 226886 Coding 11 7140 gcacctgctgggtctgcacc 44 82 1 226887 Coding 11 7145 tggctgcacctgctgggtct 77 83 1 226888 Stop 11 7660 tcgagctattacttgaacgc 0 84 1 Codon 226889 3′UTR 11 7672 gctgctggcacctcgagcta 72 85 1 226890 5′UTR 12 17 ttcacatgacaccgatcaat 69 86 1 226891 5′UTR 12 117 ctttaaggacggagggatag 0 87 1 226892 3′UTR 13 2011 tctgtgtaaaataaaagtac 57 88 1 226893 3′UTR 13 2249 tgctcgttcaacttcccttc 69 89 1 226894 3′UTR 13 2823 ctggagcatcttcttcggaa 57 90 1 226895 3′UTR 13 3186 cccgagctgagccaagtctg 64 91 1

[0296] As shown in Table 1, SEQ ID NOs 17, 19, 21, 23, 24, 25, 27, 28, 29, 31, 32, 33, 34, 35, 38, 40, 41, 43, 45, 46, 47, 52, 53, 54, 56, 57, 58, 60, 61, 62, 63, 65, 66, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 83, 85, 86, 88, 89, 90 and 91 demonstrated at least 45% inhibition of human Notch1 expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “preferred target regions” and are therefore preferred sites for targeting by compounds of the present invention. These preferred target regions are shown in Table 2. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number of the corresponding target nucleic acid. Also shown in Table 2 is the species in which each of the preferred target regions was found. TABLE 2 Sequence and position of preferred target regions identified in Notch1. TARGET TARGET REV COMP SEQ ID SITEID SEQ ID NO SITE OF SEQUENCE SEQ ID ACTIVE IN NO 143473 4 40500 gcctgcacagtgcgtggccg 17 H. sapiens 92 143475 4 45660 actccgtgggcctcaagtga 19 H. sapiens 93 143477 4 49376 aatgtggtgacgcaggtggt 21 H. sapiens 94 143479 11 454 ggtggccagtgcctgccctt 23 H. sapiens 95 143480 11 781 tgtccaggaaacaactgcaa 24 H. sapiens 96 143481 11 904 atgccaaatgcctgccagaa 25 H. sapiens 97 143483 11 1098 aggtctgctgtgccacctca 27 H. sapiens 98 143484 11 1222 gcctgcagccaggacgtgga 28 H. sapiens 99 143485 11 1399 tgcctggaccagattgggga 29 H. sapiens 100 143487 11 1963 tgtctggacaagatcgatgg 31 H. sapiens 101 143488 11 2246 actgtgacatcaacaacaac 32 H. sapiens 102 143489 11 2251 gacatcaacaacaacgagtg 33 H. sapiens 103 143490 11 2788 gccttctgcgactgcctgcc 34 H. sapiens 104 143491 11 2794 tgcgactgcctgcccggctt 35 H. sapiens 105 143494 11 3165 caactgccagaaccttgtgc 38 H. sapiens 106 143496 11 3345 cctgtgccagcatggagggc 40 H. sapiens 107 143497 11 3350 gccagcatggagggctctgt 41 H. sapiens 108 143499 11 3870 tgacttccactgcgagtgcc 43 H. sapiens 109 143501 11 4207 acctgtgagcccacatccga 45 H. sapiens 110 143502 11 4274 tgtgccacatcctggactac 46 H. sapiens 111 143503 11 4279 cacatcctggactacagctt 47 H. sapiens 112 143508 11 4507 gacggccactgtgacagcca 52 H. sapiens 113 143509 11 4512 ccactgtgacagccagtgca 53 H. sapiens 114 143510 11 4600 gaccagtactgcaaggacca 54 H. sapiens 115 143512 11 4797 cgtgctgcacaccaacgtgg 56 H. sapiens 116 143513 11 4802 tgcacaccaacgtggtcttc 57 H. sapiens 117 143514 11 4807 accaacgtggtcttcaagcg 58 H. sapiens 118 143516 11 5068 tcctcgcagtgcttccagag 60 H. sapiens 119 143517 11 5073 gcagtgcttccagagtgcca 61 H. sapiens 120 143518 11 5260 ggggtgctgctgtcccgcaa 62 H. sapiens 121 143519 11 5290 cagcatggccagctctggtt 63 H. sapiens 122 143521 11 5452 gacctggagaccaagaagtt 65 H. sapiens 123 143522 11 5457 ggagaccaagaagttccggt 66 H. sapiens 124 143523 11 5554 gctgacctgcgcatgtctgc 67 H. sapiens 125 143524 11 5559 cctgcgcatgtctgccatgg 68 H. sapiens 126 143526 11 5848 gaggccagcgcagatgccaa 70 H. sapiens 127 143527 11 6030 catgctggaggacctcatca 71 H. sapiens 128 143528 11 6035 tggaggacctcatcaactca 72 H. sapiens 129 143529 11 6076 gatgacctgggcaagtccgc 73 H. sapiens 130 143530 11 6136 gttgtgctcctgaagaacgg 74 H. sapiens 131 143531 11 6257 ccaaccgggacatcacggat 75 H. sapiens 132 143532 11 6418 ctctgctcgcccaacggcta 76 H. sapiens 133 143533 11 6523 aaggacctcaaggcacggag 77 H. sapiens 134 143534 11 6528 cctcaaggcacggaggaaga 78 H. sapiens 135 143535 11 6550 tcccaggatggcaagggctg 79 H. sapiens 136 143536 11 7129 cagcctcacctggtgcagac 80 H. sapiens 137 143537 11 7135 cacctggtgcagacccagca 81 H. sapiens 138 143539 11 7145 agacccagcaggtgcagcca 83 H. sapiens 139 143541 11 7672 tagctcgaggtgccagcagc 85 H. sapiens 140 143542 12 17 attgatcggtgtcatgtgaa 86 H. sapiens 141 143544 13 2011 gtacttttattttacacaga 88 H. sapiens 142 143545 13 2249 gaagggaagttgaacgagca 89 H. sapiens 143 143546 13 2823 ttccgaagaagatgctccag 90 H. sapiens 144 143547 13 3186 cagacttggctcagctcggg 91 H. sapiens 145

[0297] As these “preferred target regions” have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these sites and consequently inhibit the expression of Notch1.

Example 16

[0298] Western Blot Analysis of Notch1 Protein Levels

[0299] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to Notch1 is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 17

[0300] Caspase Assay

[0301] With specific inhibitors of Notch1 now available, it is possible to examine the role that Notch1 plays in cancer.

[0302] Programmed cell death or apoptosis involves the activation of proteases, a family of intracellular proteases, through a cascade which leads to the cleavage of a select set of proteins. The caspase family contains at least 14 caspases, with differing substrate preferences. The caspase activity assay uses a DEVD peptide to detect activated caspases in cell culture samples. The peptide is labeled with a fluorescent molecule, 7-amino-4-trifluoromethyl coumarin (AFC). Activated caspases cleave the DEVD peptide resulting in a fluorescence shift of the AFC. Increased fluorescence is indicative of increased caspase activity. The chemotherapeutic drugs taxol, cisplatin, etoposide, gemcitabine, camptothecin, aphidicolin and 5-fluorouracil all have been shown to induce apoptosis in a caspase-dependent manner.

[0303] The effect of the Notch1 inhibitor was examined in normal human mammary epithelial cells (HMECs) as well as in two breast carcinoma cell lines, MCF7 and T47D, obtained from the American Type Culture Collection (Manassas Va.). The latter two cell lines express similar genes but MCF7 cells express the tumor suppressor p53, while T47D cells are deficient in p53. MCF-7 cells were routinely cultured in DMEM low glucose (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. T47D cells were cultured in Gibco DMEM High glucose media supplemented with 10% FBS.

[0304] Cells were plated at 10,000 cells per well for HMEC cells or 20,000 cells per well for MCF-7 and T47D cells, and allowed to attach to wells overnight. Plates used were 96 well Costar plate 1603 (black sides, transparent bottom). DMEM high glucose medium, with and without phenol red, were obtained from Invitrogen (San Diego Calif.). MEGM medium, with and without phenol red, were obtained from Biowhittaker (Walkersville Md.). The caspase-3 activity assay kit was obtained from Calbiochem (Cat. #HTS02).

[0305] Before adding to cells, the oligonucleotide cocktail was mixed thoroughly and incubated for 0.5 hrs. The oligonucleotide [the Notch1 antisense oligonucleotide ISIS 226844 (SEQ ID NO: 40) or the mixed sequence 20mer negative oligonucleotide control, ISIS 29848(NNNNNNNNNNNNNNNNNNNN; where N is A, T, G or C; SEQ ID NO: 146) or the lipofectin only vehicle control was added (generally from a 3 μM stock of oligonucleotide) to a final concentration of 200 nM with 6 μg/ml Lipofectin. The medium was removed from the plates and the plates were tapped on sterile gauze. Each well was washed in 150 μl of PBS (150 μL HBSS for HMEC cells). The wash buffer in each well was replaced with 100 μL of the oligonucleotide/Opti-MEM/lipofectin cocktail (this was T=0 for oligonucleotide treatment). The plates were incubated for 4 hours at 37° C., after which the medium was dumped and the plate was tapped on sterile gauze. 100 μl of full growth medium without phenol red was added to each well. After 48 hours, 50 μl of oncogene buffer (provided with Calbiochem kit) with 10 μM DTT was added to each well. 20 μl of oncogene substrate (DEVD-AFC) was added to each well. The plates were read at 400+/−25 nm excitation and 508+/−20 nm emission at t=0 and t=3 time points. The t=0×(0.8) time point was subtracted from the from the t=3 time point, and the data are shown as percent of lipofectin-only treated cells.

[0306] It was thus demonstrated that inhibitors of Notch1 induce caspase activity in all three cell lines tested. The Notch1 inhibitor ISIS 226844 caused roughly an 82% reduction of Notch1 RNA and approximately a 2.6 fold increase in fluorescence (indicating apoptosis) when administered to HMEC cells at a 200 nM concentration. In MCF7 cells, this Notch1 inhibitor reduced Notch1 RNA levels by approximately 65% and increased fluorescence (indicating apoptosis) by approximately 2.6 fold (200 nM concentration). Similarly, in T47D cells, Notch1 RNA was decreased by approximately 75% and increased fluorescence (indicating apoptosis) by 5.5 fold (200 nM dose of ISIS 226844).

Example 18

[0307] Cell Cycle Analysis

[0308] Cell cycle regulation is the basis for various cancer therapies. Under some circumstances normal cells undergo growth arrest, while transformed cells undergo apoptosis and this difference can be used to protect normal cells against death caused by chemotherapeutic drugs. Disruption of cell cycle checkpoints in cancer cells can increase sensitivity to chemotherapy while cells with normal checkpoints may take refuge in G1, thus increasing the therapeutic index. ISIS 226844, an inhibitor of Notch1, was tested for effects on the cell cycle in normal HMEC cells and cancer cells, both with and without p53. 72 hours after treatment with antisense inhibitor, cells were stained with propidium iodide to generate a cell cycle profile using a flow cytometer. The cell cycle profile was analyzed with the ModFit program (Verity Software House, Inc., Topsham Me.). Neither lipofectin alone nor a panel of negative antisense controls perturbed the cell cycle. However, it was found that ISIS 226844 induced apoptosis in all three cell lines, as measured by an increase in the percentage of sub-G1 cells. In T47D cells, the percent hypodiploid cells (indicative of apoptosis) was shown to increase from approximately 0.5% for lipofectin control-treated cells to approximately 4.3% for ISIS 226844-treated cells. In MCF7 cells, the percent hypodiploid cells increased from approximately 0.7% (lipofectin only) to approximately 2% (ISIS 226844). In normal HMEC cells the percent diploid cells increased from approximately 1.6% (lipofectin control) to approximately 2% for cells treated with ISIS 226844. This increase in apoptosis was dose-dependent.

1 146 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial Sequence Antisense Oligonucleotide 3 atgcattctg cccccaagga 20 4 55001 DNA Homo sapiens 4 tcctctcctg gggcgctgac cccaagatgt taccccaggc ctgcagaagt gaggccagat 60 tctgggagga cgcaggcgag ggaggctccc agcagcagct acaggggcgg gacaggccgc 120 ctgcactggc tgtttccaga gtgctcagca tttggcaaga ggtgtgccag gaaaggcttg 180 ctggggtcag gtgtgagatt tgtcctgctt tctgtgcctg cctctgccac catgctctgg 240 ggtttctgca gccctccccc caggccccca cagtcctagg tccctcccag cctttcggtc 300 tccccgcagg gcaggcttag ccctcctact ccaggagggg cgccgggaca cgcgccttct 360 gccatcgcac tcaccaccct gtgttggggt ggggaggggg cgctgtctgt ccctggcacg 420 aggccgtgaa ctttgcgcag ggactatggc aggcattttg gactcctggc gcttaaccag 480 gcttggcaca gggtgcccgt gcctggcatc gtggtggaga aataaatcag ccggaaggag 540 cacgtgggaa gggctgcgcc ggcgccagcg gcagatccgc ccgacccgtt tgtgctttct 600 ggggccacct taggcaggcg gcgcccgggc aggaggagga cggtgaccga ggagcgtgtc 660 gacgcgggtc ccccttgggg gagcggggca caatccgtcc gcgaggcact ggtcccggct 720 gctccctcgg ggcgcccgga gtcccctgcc cagaggccgc ggcgccccca tccctcgcgg 780 tcaagtgctc gggcaatcac ggccagggat gtctggcggc gatcgatcct ctggacgcct 840 aaagccgcgg ccacggggcc ctcgggaggg agtgaagccg ctgggctagg ggcgcacaca 900 cggctaggcc actctcccag agccctgccc cgggcccggg gtcccccaac gtggcctcag 960 ctgctccccg cccggcccag cgcacggtgc acacggctgt ccgcggcctc gccctcccca 1020 ttccgccccg ggctcctccg cttattcaca tgcaaatttc agtcgccagt tgtcgccgag 1080 cgcggcaacc gccagagccg gatccttccg cagccccggc tcaaactttt ggcctctgaa 1140 aactttcaaa cgagaagtag tcccaggcgc ccgctcccga cccacgccgc gccgacgggt 1200 ccctcctccc cggagaggct gggctcggga cgcgcggctc agctcggaga ggcgcaaagg 1260 cggacggtgc gtgcgggagg aggtggtcgc cgagggggcc gagggaccgg cggtggtggg 1320 gccgggcgga gcggggccgg cggtggcgga gcgcacctcg actctgagcc tcactagtgc 1380 ctcggccgcg ggagggagcg caagggcgcg gggcgcgggg cgcgggcgcg ggcgcgagcg 1440 cagcgaagga acgagccggg cgcggagccg ggcccggggg cccgcgagag cacagcgccg 1500 ccagccagcc ggggaagaga gggcgggacc gtccgccgcc gccccgggac cgtacgccgc 1560 gcgtgtgcgt cccagccccg ccggccagcg caggaggccg ccgcccgggc gcagagggca 1620 gccggtgggg aggcatgccg ccgctcctgg cgcccctgct ctgcctggcg ctgctgcccg 1680 cgctcgccgc acgaggtagg cgcccaccca cccgcgagcc cccactttcc gcgccctttg 1740 gaaactttgg cggcgcccgg cgcgcgcgcc ccacggctgg gagcgggcgg cggggaggcc 1800 agcatggaga gggaaaagcg ggcggcccgg ggcgtggggt tctggagtcc cgggatcagg 1860 gaggaccgac cttccccctc gatccccccg tggaggcgga ctcgcgccgc ccgtgcctgg 1920 agccgagtta ggaggccggt gtggggtgct ggggccccgg aggccctact ccgggcccgc 1980 ccttcacccg ccgcgcgtgg ggcttgccgc cggtcggccg ggcgggcggg ctgcctacta 2040 tttttcgatt tgaatagagt cggttttggt ttcctgttgc ttctccgggc catttatctt 2100 ctttcttctt cgcctctggc ccacgccggg gcggatgttg gggcgcggag tgtgggctct 2160 gcggcgccgc gttcgccttc actgacccgc gcggccgggc tgggtccccg ggctcccggt 2220 cgccccgccc gccggtgccc cccagcccgg ctctcagttt gggggagggg ttgcgtaaga 2280 agccgccgcg cccgggggga ctgaactttc cttttgcttt gcggagttga agtttggaaa 2340 gcttgggggc ggagagcggg acgcgggtgg ggggctctta catttctccc cgccgcacag 2400 cgagcggggt ctctggggaa tcgagtgatt aatccactct ttctccgaga gttggaggcg 2460 agaattatct gtcctcttcc agaaagtgcg gctctgtgtc acacccccct cccccgtttc 2520 tcagccccgg taagatgggg agggaggggc ttgagtaatt gatcccttct cgagatgggg 2580 tcgaattcct tccgaatggg ggaccttcat ccccctcctg tgggtgtatg ggggctgccc 2640 tggagatgcg cgcccgcgga ggcaggtatt gggtgtcggc ggaggcgggg ccgcgtcccc 2700 agggtgctgt cccggtgccc ctggaggcgg ccccgactcc acaatgggcc gctctgattc 2760 tgaggcggag gccggcgctt tgttggcggg cggggtagcg ggcgagcagc tgtcgcattt 2820 tcccacgggc gggagctgag tgtggccacc ccccctcccc ccgtccagct ctgccccctt 2880 gcagaacggg tttgaacaga ggcaatctcg gcggctggat ggggggccct gccctgccag 2940 actcctcagt gagcctccag ggtggggggc aacgctttgg agagtagccc actttgtctc 3000 tgcttttccc ccactgcgtc caggcggcac atcaggccac cccaccctgc ttcaagcagg 3060 acgtgttccc tgctgctccc cttcccccca tttctgacta cagaactctg ggcagaatgt 3120 tgacgcccct ttggtttcat ggaggggctt cctgcggacc cgtgccccag caacccatga 3180 tactgagcag agtgcgtccg gggtaggggg cggacgatgc cccttggctg ggtgcaggtc 3240 cccgccccca gggtaaccag ggccttcggt ggtggtggtg cgggtgagac tgacctctct 3300 tctcctgccc ctgcctaggc ccgcgatgct cccagcccgg tgagacctgc ctgaatggcg 3360 ggaagtgtga agcggccaat ggcacggagg cctgcgtgtg agtaccaccc ctgcgggacc 3420 tgttgctttg tcgagggcag agcccctgcc ttctgcagcc gcgcagggga cagaacacta 3480 ggccattgtc ttctggagat ggcccagagt ctgggcacgg tcacgtgctg acttttattg 3540 gacaaagtct ggcaattact taatcaccag caattatgct gcgtgtggag ctggtctggc 3600 tgctgagggc ttgggcactc ctccgtgggc cacctcaggc tccggggtca ctaagtggga 3660 gtcccagtac cccaattctt ctttaagtcc ccaagaaaca gttgatctgc caggggaagc 3720 tctggaccat ttactgagag gcccagcccc caccaccctg ccaacttggg gcgcctcctc 3780 tgggcgggtg atgcctcctg ggcacaggtg atgcctcctc tcgggggtga tgcctcctag 3840 gtgggtggtg atgcctcttg ggctggtagt gatgcctcct ctggcacagg tgatgccacc 3900 tcctgggcgg gtgatgcctc ctctgggtgc gtgatgcctc ctgggctggc ggcaatgcct 3960 cctctgggca caggtgatgc cacctcctgg gcgggtgatg cctcctctgg gtgcgtgatg 4020 cctcctctgg gcacgggtga tgccacctcc tgggtgggtg atgcctcctc tgggtggtga 4080 tacctcctct aggtgggtga tgcctcctct gggtggtgat acctcctcta ggcgggtgat 4140 gcctcctcta ggcacaggtg atacttcctg ggcaggtgat gccacctccg ggtgggtgat 4200 gcctcttcaa aggaaattga ctcttcaaaa tcggacccgc ctttgggctc tctgtttgcc 4260 ccagctccct cttcccggcc tcaggctggg gctggggaag agaaggtgga actcatgttt 4320 tggcgtgggg tgtggggtaa ttattgagtc tggtttctgt cctggcatct gcaaactctg 4380 acctcaaaat cccgtggccc ttctggtctg ccagcctcag atgtaaaatg ggcactgagg 4440 gggccgggaa gtcgtgcgga accgctccct cctgcctgcc tgtgagtgcc catcagccgt 4500 ggggaactgt tctgctgtgg ccgatgccct tcaacttgag tccatccctg atgagaataa 4560 tgaggaggtg tttggtgtct ggaaaaagag ttccctccgc cccccaggag ctacagatga 4620 agaaatggct ccccagccag tctgcgcgcc ctcattccaa ctgcagaagg tcccattccc 4680 ttgacaaccc cagcccggct cctgcctgtg ccgactgccc agcctgtggg cagcaggccc 4740 cctccctccc cggcccccac cctccccaaa ctgaagccaa ttaagcaggt ctgtgagcag 4800 tttaatggac aaagccgatt gtgtctttgt cagacactaa tgaatgcgca ctactttttt 4860 tttttttttt ttttttgctt tttggggccc ccttctccct ctgcagaagc cgcacggacg 4920 cttgatgccc taaatcttgt ttcctttcat tcagagaccg acagctggga tccaggggaa 4980 gtgcagcagc tattgttggg ggaggcggat taatgccgtg tgattaatta tgcgtggctt 5040 cccagaatgt ataaccccct tccccgccac tgcgctcccg gctcggactt tgcctctcac 5100 ccattcgggg ctctgggaga agcttcttgc tcctgcagag gactttggga gggtgggagg 5160 aacaccgtgc tgggaaaatt gggggcctgt tgcttcttct cagtggcacc agggagtagg 5220 cacagcctgg cccacagggg tctgggactg aggtctccct gggccagagc gctgtgcttt 5280 ggggtacaga ttgctgcagc tccctggtgt ctgggttaaa accctgccac agggccgcaa 5340 aacactggtg tcatgggccc actttcccat tccccaaacc tgggatcccg gtgagctgtt 5400 tccagcccaa ggcaggccgc cgaccatggc tttggtgcct ggccttgtcc cctttgctgg 5460 ggcctggcgc cccctccttc ttggcgggaa gcctgcttca gtttggcctg acaggcagcc 5520 gagctcagcc cacagagacc ccattgtgca caccccacaa gtccaacgag aagatgggac 5580 ggaagcgcgt cctcaggctc ctgcttctgg gccgcagagt tggaggccag ggccacagcc 5640 gagcccggct tgtggaggca caggtcaggg ctcacacagc agtctggggg acattgcagc 5700 cgcttcggag aaggccccgg gctccctccc tgggcttgag ccccacccaa catacacccc 5760 tcaggtgcct ctgccttccc gttcctgccc atccttctca gaaggcccag gaggtctgtc 5820 caggagttag ccagggcacc tgcaggaggc ctggctcccc cagcaagggg agaaacccac 5880 actgtttctc gaaagagtct ggggtgcagg ctcttggagc cctgccagcc tgagtgggaa 5940 tcctggcccc gtggtatcca gctgtgaccc cggaaagagc tttggctttt ggtgccattt 6000 gctgcctccg ctgtagaagg ggcatggtca tgatgggcgt cacagtgcct ggctcactag 6060 tactgggtga atcatttctt tccatcccaa agagaaggaa ccttcaccgg aagctgccaa 6120 tcaggtggtt gcagggcccg ggattggaaa aagtggtttt gttggtgcaa aatgccttcc 6180 caggaggtca gagtgtcgcc gtggcctggt cagccagtgg ccaggtagaa agcacctggc 6240 agcacatgct ggtgggcgga ggatggttgg tctctgggca gctgctgtgc gaggtgtggc 6300 cgtggtggct gtggtcggag atggtggcct tctgagcagg cccagggggt cactgtggcc 6360 cttggctctt ctctagcccc ctcatcagct ccgtcagtgt gagcaagggg tactgaagag 6420 ggctccacct tttttctctt tgttttgagg agagctggga agagctgcag ggaccctatc 6480 tgtctgggtg agactacgtc cttggtctga aacgctctcc cagggctgcc tgcgtctggg 6540 ctgggcgttg aggcaggggg cagctaagat gttggctgct gacggcagag tcagaatggc 6600 acggggcgtc ttccaggagc tgggagaaat gggggtaggc tgtcacaccc agcacctcca 6660 ggaggatgtc cagctgcctg ctgggtggtg cgctttagct ttggacaacc tcgtgtctga 6720 ggcctggcct ggccactagc tctgtgacct tgggtcatca ggtcatgcaa gcctcagttt 6780 ccccagcatg tcagccgggc tgatacacgc tggatctctt ggtggtgtgt gaggattgca 6840 tgagaaccgt gtggagaggg cacctggtca gcgctccttt cttccccgcc ttctccccgt 6900 tggctgacag tgctggtttc cagagcctcc ttcctgaatg tttaattgat tgacgagtga 6960 attcagagaa tcctaaaggt gctgcctggt gggctgggcc tggcactgcc tggggaggga 7020 cttgccggct ctggggaagt ccatctccca ggatggctcc agctccgggg aagtccatct 7080 cccagggtgg ctccagttca cagacacgtt tgagcgcctg ctgtgtgctg cgttctgctc 7140 caggccaggc ccccggtggg gacagggcag acatggcccc tgctcctgtg gagttcactt 7200 cccagcaagg cgtgagagag gcacctagct cacatgtggg acatgttggg ggtgtgccgg 7260 cccggtggtg gcctggcata gctcccggca gccccgtaaa gctgtctgga tcaggcaccg 7320 ataagtgaga agaagagacc cagagaagtc gccatcagcc ccagggtcac acagcagtgg 7380 cagaattcct actagccctg cccctctcct tctcccaagc gaatgtccct aaacacagcc 7440 ccagccagcc tgagctgccc cgtcatttcc cgactacaag cggactgggg gcgtggcttc 7500 cccttaaaag aagaggaagg aggctcaggc gggaagtgac ttggccctgc agccggcctg 7560 ggaggctggg gagggacggg gtagctcctg tcacccggtc tggctctttc cattgagtca 7620 cctgcctcgt cttgggcgtg gccaggggag gaacaggttg attctcctcc tcatgctgag 7680 ctgagcggaa aggcctgtga caggacctcc tgtttatgca gaacctggtc ttcaagggcg 7740 ccagggttag aaaaacatgt ttaaaaacat gcagcatccg aggtgggcag atcacctgag 7800 gtcaggagtt caagaccagc ctggccaaca tggcgaaacc cccgtctcta ctaaaaatac 7860 aaaaattacc caggcgcagt ggcgtgtgcc tgtaatccca gctacttggg aagctgaggc 7920 aggaggatcc tttgaaccca ggagacggag gctgcagtga gctgagatcg tgccactgca 7980 ctccagcctg ggtgacaaag tgagactctg tctcaaaaaa acaaaaaaca acaaaaaaaa 8040 ccccacacag tgtcaaatag aaagtctcac ctactcagac tggggagaca tcaggaacag 8100 gaaggcctgg ggcggagact ggggtgggag cctggagtgc tctgaaggaa gctggctcct 8160 tggggcaggc cctgcctctg aacagcagcc aacatgtttt ccattgaaaa gaataataat 8220 agaaaaagag agagggagaa agaaaacagc aaccctggca ggaagagggt agggcagggc 8280 agacagtgca ccaggggaag tgcttccaca gggcagagct gggtgcgggg acccccagat 8340 gcgggcggca aactggagac cccacaggag ccgggactgg cttcattcct cattccctgc 8400 acctgtgatg ggcagcagcg gccaccggtg cccactgggc agttccaggc cgggccgagg 8460 gactgcaccc ttttgctgtg gggaggggac ctttcctgtt tcatgtctcg tctataaata 8520 gtgaaaggat gtggctttgc agaatcgatt gggcgtctag cttgccttcc tcaataatgc 8580 agcgatgaca gctccacact caggacgccg ctgttaccca gcctcgtaaa agccctggca 8640 gacggccgcc caccctccag gtgtgccagt gcagttttca tccccagcgc acaggtggga 8700 aagctgtggc tgcgagggcg tggggatctg aggcaggctg gcgtcagctt tcaccttcat 8760 gtgcaggcat atccagaact ttccggatct ttccccgggc tgaggaggaa ataccagcag 8820 ctttaacgag cggctggctt tggggcatca cacctgtggg ccgggctgcg tttgacggcc 8880 tcgagtttcc caggcagcgc tggcgctttc tcctgtgggg cccggagctc cgaggggctg 8940 tgaagcaggg ctggctctga tttgctgtgc tccccacact gtcccctccc tgggcctcag 9000 tttccccatc tgtatgtgat tccccttcca actacagcca gcgggaccct ggggcgggct 9060 gaaggcgtgg tggtggctgt gggccctgcg gcgaggcctg cgctgggctc ggtggtcgcc 9120 ccagctgggg aaggggcagt ggctgcaggg gtgggagggt ggagggaagc ccttttccaa 9180 agttgcctgg ttgggttctc cttgtggccc tgccccaccc caccctgctc cctgggagca 9240 aagggagcta aggactggtt tggggatgga tatgtatatg gagggattct gggtgatgac 9300 ttattcatat tacaaaattt gccttttttt gtttgttttt tgagatggag tcttgctttg 9360 tcacccagac tggattagag tgacgcagtc ttggctcact gtaacctctg cctcccagat 9420 tcacgcgatg ctcctgagta gctgggacta caggcacata ccaccactcc cagctaattt 9480 ttgtattctt agtaggggca gggtttcacc atattggtca ggctggtctc aaactcctaa 9540 cctcaggtga tccacctgcc ttggcctctc aaagtgctgg gattacaggc gtgagccact 9600 gtgcatggcc acttttttgt tttttgagat ggggtctcac cctgtcacac aggctggagt 9660 gaagtggtgg gatcacggct gactgcagcc tcaaactcct gggctcaagc gatcctcctg 9720 cctccgcctc cagagtagct gggaccacag gtgtgcacca ccatgcctag ctaattttaa 9780 atttttttgc agaagcgggg tcttgctatg ttgtccaggc tgaaaatgtg acttttgaac 9840 taaagcatgc tgttgcttga catctagtcc attcacaagt tcattcaacc actatctagt 9900 tccgggactt tctcactgca caaaacagaa actgtaccca aatactgtca ctctgccctg 9960 actgccccag cccctggcaa ccactaaccc gctttctgcc tctgaattcg cctcttctgg 10020 acgttgccta tgaatggaat tgtgccatag gtggtctttt gcccctggct tctttcactg 10080 agcgtgatgt gtttaaggtt catccatgtc acgcgtggac tagaactgca ttcttctgag 10140 gactacattt tagcctccag aagcatcagg gactggccag aggtggaccc cagcatccca 10200 gctcccagac agtccagagg aggcaggtgt tgggagggcg tccggggtgg cttcctggaa 10260 gagatggcgc tcactctggg ccggtgcagc cacccgacag ccacagagcc ccctagaggg 10320 agtcctggca tattcccagg tgaacctgta ggatggactg gcgtgcccgc ggcccctgtc 10380 ctgctcagga ctccgagaca ggctcacaac ctgtccctgg gcctcactca tgccagccgc 10440 caagctgttc agcatccctt agcccccagg aggcacacag tggggccgcg gtcactgagc 10500 gtccacacgc ctggtgctgt aagaatgcaa agtagagggt ctttattgag catctactac 10560 atgctgaacc ctggggtatg ggtgggcacg gcagatgcag gcccccctgg gcaggggagg 10620 agcatgcccc ttaagaacca ggccacagga tggacgggct tggccacaag ctaggggtag 10680 tcactgcagc cttcatggcc tcctgtgtgc aaagtcagtg ctgtgggacc ccatctgggt 10740 gcacctgctg tggctttcaa gtgaggccca ccacggagca gcttccacgc tggctgggac 10800 agggtcagcg gcagggagct ccagaccaaa ggagcggcgg tggctggggc agcggggggc 10860 agtgaggctt tggacacatc catggggcgg gcaggcatct cagcccccac ccgcccagtc 10920 tgcctgctcc ggggccttcc cttccctgga cagagctctg tgtgattggc tccaggctgc 10980 ttctccccct ggaccctcag ggtcagctta gagtgactgg ctatgccctg gcctagatcc 11040 aggcccgtgt gacaggagag agctaggaac ctggcccctc actctgagac acagatggtg 11100 tcctctccaa gttgcccttg ggacggggaa gcgccatgtc ccctgcaggg caccaggcca 11160 gccagtggcc cttgacagcg aggccctgtc tgcaggcagc tgggggactt ctccctgtcg 11220 ggaggatgcg tgcgtgttag catcggggtg cctggttgcc ccaggggtgg tgcagggcgg 11280 ttataggcgg tcctagcatt ttggggttgc tttcagtgta tctccgagac ctgtggaatt 11340 ctgggaggag aaactcccca gcagtgtcct cagtgcgccc ccctaccctg tacccgttgc 11400 tcctgggagg gatcctgagt gctccccccc acccaccatg cacccattgc cccaggaggg 11460 atcctgagtg ctcccctctc cacccaccct gcactcattg ccccgggagg gatcctgagt 11520 gctcccccct ccacccactc ccaccctgca cccattgccc ctgggaggga tcctgagtga 11580 tcccccctcc acccaccctg cacccattgc ccctgggagg gatcctgagt gctcccctct 11640 ccacccaccc tgcactcatt gccccgggag ggatcctgag tgctcccccc tccacccact 11700 cccaccctgc acccattgcc cctgggaggg atcctgagtg ctccccccga cccaccctgc 11760 acccattgcc ccaggaggga tcctgagtgc tcccctctcc acccaccctg cacccattgc 11820 ccctgggagg gatcctgagt gctcccccct ccacccacca tgcacccatt gccccgggag 11880 ggatcctgag tgctcccccc tccacccact cccaccctgc acccattgcc cctgggaggg 11940 atcctgagtg ctccccgcct accctgcacc cattgcccct gggagggaga cttctgtaga 12000 ggcctcacct ctgaaccccc cactcacagg ctcaggggcc cagggctggg ggcgtcctgc 12060 cgtctacact ccagattgag cccgcacggt taaacagggc cctctccgca cgccctgcct 12120 aatgcaaagc ggtggcttcg ggtgggtccc caaggggcct tgtgaaccct gggggcctgg 12180 cccctcccac ctggctgcac ccgctgggcg ggagatccca tcctccgagt cctcagcccc 12240 tcagcctcag ctgcagccca gacaccaggg ctctgtgggg cgggggtggg gcagcggttc 12300 cctcaggtct gtggggctgc cctgctgtgg ttcctgcaat ttcctggaag gaggtctggg 12360 gcctggtgtc cagtggggcc tgtggggacg gactcggggg ctctgtgggg agggactggg 12420 ggctctgtgg ggacggactt gggggctctg tggggaggga ctggggggct ctgtggggag 12480 ggactggggg ctctgtgggg agggactcgg gggctctgtg aggagggact ggggggctct 12540 gtggggaggg actgggggct ctgtggggag ggactggagc ctggtgtcca gtggggcctg 12600 tggggacaga ctcgggggct ctgtggggag ggactcgggg gctctgtggg gagggactgg 12660 agcctggtgt ccagtggggc ctgtggggac agactcgggg gctctgtggg gggggactgg 12720 ggggctctgt ggggagggac tgggggcctg tggggacgga cttgggggct ctgtggggag 12780 ggagtggggg ctctgtgggg acggactcgg gctctgtggg gagggactcg ggggctctgt 12840 ggggagggac tgggggctct gtggggaggg actcgggggc tctgtgggga gggactgggg 12900 ggctctgtgg ggagggactg gggggctctg gggagggact ggggggctct gtggggaggg 12960 actggggggg ctctgtgggg acggactcgg gggctctgtg gggagggact ggggggctct 13020 gtggggaggg actggggggg gggctctgtg gggagggact gggggctctg tggggaggga 13080 ctgggggctc tgtgagaggg aactgggggg ctctgatgtg ctgggcgtgc acaggggagg 13140 ctttgcctct gacatccaca cctgcagttc ccaggcacac acagcggccc catcggggtc 13200 ctcgcaatca cagtctgtga gtggccacta ccaagggcgt ctccggatca ggccaggccc 13260 agatgctgcc cggacccaac ccccacagcg cctgccgcca cagtagggac caatggccac 13320 agaacactca cccggcctca cttgcacggg gctgttcagt ctggtgggca ccaggtgggc 13380 gctgttctcg cccagtggga ggcctgccag ctccttggtt gctagttgag atttttcccc 13440 aagaaatagt cgtgttgttc cttctgctcc gcctggcact gaggttggtg acacgccccg 13500 accttgctct aattggcaga tgagaatttg tcatcagatg tcgaccctgt ctacgcagtc 13560 ccctggcctc ggagtccatt gtgcatctct gaagggcttg aattggttgt ttaaagctgg 13620 gtaaatgccc ccttgacatt ctgttgacac tgtcaatttg ctgaacaaac tcttccacag 13680 gtagcacagg aggccagtct cgccccagcc tgagccaagg cccagggagg caggtcctgt 13740 gtggagtcag cctttttgtg agtgcagcgg ccctgcagga tgcaccgggt ttgagaagtg 13800 gactcccccg tctcccgcca catcccaaaa gccctcagcc atgagggcag gacgaagccg 13860 acggtcgccc tcgtgccgag atgacaaccg ggcacagagg cggtggctcc cagcttcctg 13920 ccctcttccc cgtagtgggg ttacccagcg tgactcatgg cccgagccac aggcacccgc 13980 ccagggagcc acggcgggga gacctggctt tattagagat gcgttcgtgc ctcatcaagt 14040 ccaaaggagg aaactggcgc gtccctcact ttccctgaaa caggctcccc cacagcccaa 14100 gcggtcaggt taaggtgcat ttgcagacag atgccctagt aggaggctgg agcttttggc 14160 ccagaatttt ccctgctgta ggcccatggt caccgagcct gcccgggggc tgaggccctg 14220 aggaggtgcc attgccccac ccaagcctca gacaagtgtc ctgccctcga ccctgcggga 14280 ggcagcagct caggccctga ccctggccag agagggcagc tctctgaggc tgctgtggcc 14340 gtggcaggca ggggctgggc ttgccgggcc gtcgagtggg cacagagact atctgggaag 14400 gaggtgggcg gtgagagctg actccccgca gccgggggac agatgagccc cgtgccgcac 14460 agcagggccg cgtggtgggg ccccccaagt tccgctcggt ttcagctgtt gacgagcgga 14520 aacacacagt tactggaaat gagaggtttg ggggagccgc caggggttga acatgtgtgg 14580 tttccctgac ggcttttcag taagagatgc ttgagctcag gctgggcact ggctggtgcg 14640 cggagggggc tgggccagag tccccccctt gggcctccac tctccatcct tatagaggga 14700 cctgggttgg tcccagctcc cggggtgcag cccctgggtg gcccctccta cttccccttc 14760 agtggtcaga aggaggctgg cctcattcct gtctgacgga gagccaggtg gggaggccgg 14820 gcggggctga gtgtccaggg tccacatggt ggcggggctc aggccgtggc gaggtcctgt 14880 cctgggcctc cagggtctgg cccagcacag agctctgcag ctctctggtg gacgggcctg 14940 gctgggctgt cggcagccct ggggccaggt gggcagtggg aatggaggtg gaggcaggtg 15000 ggtggctgca ggtgactgga aggcttggct ccagcctgca ggggctgcag tccaggccgg 15060 ccacccccac gagaccttcc cccttccagg tgtggggtgg cactgggtca acaacacagg 15120 ccacggactt ctttccccca cgtgggccgt gcgaccccgg gccgccacca cctctctgag 15180 ctcccctcct ccttaggaga tgggaagcgg ctggggccat gaagtgtccc ccagggctcc 15240 tgacagcccc agctgccgtc ctcgtggctg ggcgactgcc gcgaattgtt gcgccttctt 15300 ttgagttggc tacggagtca tggcggctgt gggaggtgta gctgggtctc gggcggcacg 15360 cccggctccc ccggggatgt gtcatctgtg tcatccaccc tggagcccct tcctggtgct 15420 gtgggggccc ctccaggaag gtctctccca gaggccagcc ccagccacag gctctgggga 15480 gactgtgtgg gatttgcatg cagttgtgtg ctatttgatc gaggtcatgt gtgggatttg 15540 ggcgaggtcc tgcgggattg ggtgaggtag tgcgagatct ttgggcgagg ccgcgaggca 15600 ctgggcgagg ctgggcatta ttcagtgcct tggaggggca gaagttttag gacctgccaa 15660 gtccagccca agaatcccca actttcccca cagggaaagg taccccagac agtccccagc 15720 ctgtgccagc tgtcttggga caccctggtg tcccccaggt tgtggcactg caggcccctc 15780 ccccaggtgc ccctccttat gggcccctcc cagggctgca ggcacctgcc tggatcccac 15840 ttcccaagtg tggggtaatc ccaggcgccc ggtgatcacg ctgggcccgg gcagagtgag 15900 gctctgaggg ggtgccaggg gcacagtggg ccagtatgtg agccgggctg ggcgctggga 15960 ctgggccggc aggcgagcgg tgttccgaga ggaggattcc tgggctcgga ggcccctctg 16020 tgcctcgtgt ggtcagagag cagggagccg gcgggtgtga tggggatgag gtgacctctg 16080 gggccaccac tgaagccgcc gtccatctgc ggccgaatcg ggaggcacac agaggtgtcc 16140 tggtggtttc ccgggacagg cgggaggagg cgcaggaggg cggcgtctgc ccccgggatg 16200 ccaggagtgc tcctccggca gcgtgggctt cggcttcgtc cctctttcac cagtgccgac 16260 gccccgcggg tgctgtgggt gaaggggcat cggtgccctc ctgctgggcg ggaggaagga 16320 aggggagcgg gaggctgggt ctctctcgcc cagggctctg cccccaccac cctagggttt 16380 tgtgtttcgg tggagctgta agaatgcttt gtctgctcca gacttcccgg tcctgggaga 16440 tcaggtggtc aggcaaaaca tccagaccgt ctgggtgggg ctgggggtgc gggggacacg 16500 cgccttcctc ctctttccca gggagcttct gccactcctg tgccaccctg gggagctgcc 16560 ttgccctgcc ccgctctggt ccccatcagc aggtgtggtt ctgagctgcc ttcagatccg 16620 ggtggtgtgc tgggcatagc tgccccgttt tctcacctgt gaaatgggcc cattacacgc 16680 ccccagggcg gttgtggggg tcccatgaga cagagcttgg aaagcccgag tgtaggtcag 16740 gccaggaggg ttgtgggctc agtttcttca ctgctgggct ggtgtgtgcc ggacagatgt 16800 ctcgggatcc cttccctccc tgcagcattc ttaacaagga cgctggacat ggagtcagca 16860 cctccgggcg actgccctgc tggctgtggg gctcactcca gaggagaaca ggagcccctc 16920 cggggagccc cttcctgcca cccccagcga caggatctgt tcaggagcag tggtgggagg 16980 ttgccagaag catgtctggc gctggcccgg agcgcagcct gtgatgcccg acttcattca 17040 tggagtgtgg tttgagccct ccagcagcgc tgctgtgggc ctgtccattg ccttccctgc 17100 ctctgagcca tcgtggggtg cagggctggg ctgggtcttc ttgggagcag agccccggcg 17160 tgtcatgacc atcttggcct ctccaactca agaggtttgt ccacatgggt ccctaggggc 17220 cgttggggtg acccagggca ggagacacat gcctgctttt gggggatccg cgtgggaaat 17280 tcccgcagtg gagcagcagg tggggctcca gcaggcgttt ttcacactcc aaacagcctg 17340 cgggggcact ttggtgacac ctagttcctg gggtctcaga gcctgctggg ctgtgtcttg 17400 gcattttggg gaggggctgg ccacatgccc ccagagtggc caccccagct ccgccatctc 17460 cacgggcctt ctgtcggtca gatgcagaca gccgttccca tgtcgggtga gggcattgtt 17520 ttcccggctg cgggaaaggt agcttcccgg gaggacaggt tctgtcccca tcccgcctcc 17580 cacacaaagg gtttccccgg agccagagga ggaaacctgg ccttcctgca cttcctcctt 17640 cgcacatttt aaaccatccg aggatgcaac tgggggaaaa gatttgcaga caaaagagct 17700 gggggtgccg gcaacagctg tttgggccgg aaagcctggc cccggctcag accccggctg 17760 ggtccctgcg ctgtggcggg ccggccgtga cccccgcccc acccaactcc gccccctgct 17820 gcccgctgtc tccggagcgg cggctgtttg gggctggctc cttttctggg cccccacctc 17880 cctgaagccc ccactgcccc tttcctcctt cctggggccc tgggagccag ggcagggccc 17940 ccgtgcagct ttcagcagct ccagcccaac ggctgcaggc gctggggaag accacagctc 18000 aggtgggacc gcaggtggtg gctggaggca gccatcccac atcgcagccc ccaatctcgt 18060 ggttttgtgc cccaacggtg aacaggcctg gtggtgctgg gctgtcgtgg gccccttggc 18120 cccctcggtg cctgcatccc tctctcctgt ccaagatggg gaaggaagcc gggagcagaa 18180 agggaagaaa gacccagcgg ctgcagggcg cctttcaggg cctcctagcc accgcagagg 18240 ctccagccag ctgtttgaac aggggttggg acacagggtc ctggcaggtt tgaggagccc 18300 ggggccttcc ttgctatagc tctttgttct gggggatttg gggaggccag ggaaggggct 18360 gtgagcaacg aggttccagc atcttcccaa gcctcctcca tcccccaggg ctgggctgct 18420 tggccgttcc agtgggagag gacccgaggt gggaccccga gccccctgcc cagctcattt 18480 cttagcagcc ggtgcctcct ggaaggtggg tggtgcccac tgggattggg gaaacttggt 18540 ctagcctcta cccaagggag gggctgaggt cccaggactc cccctgcccc agaagccttg 18600 gtccgggcta gagctgggct cgttggcctc atggaagcct gccttgggaa gcatccccac 18660 ccaggtctgg ccttgtctcc ccgtctccag tagggggtca cagtgtcccc ccagccagtc 18720 cacatatggc cccatttgct tgggcaggcc gatgggtggg cctgtctgtg tggcccaggt 18780 cacctggtgg ggcccactgt cagtggcgta ggtagaaggt ggtcagcctt gctgggtctt 18840 agcagagcac ggcaggatct gggtctgggg agggcgtggg ggtgcccctg ccatcacagg 18900 agcaggcaga gtgggagccc tagactcccc tctgggcaga aagccccttg aggctggggg 18960 caggtcctgc tggtgaggga ggggtcccag gggcaggggc accctcaggc tggccgccta 19020 gcgattggct cctgcaggac gtggccacgg gctccctggg agcagcagct gttgggggtg 19080 tctggggaga agacccccat ttattgccct gtgagggacc acactgctgc caggggaccc 19140 ttgggcttct gtggcaggac agtgggccgg gattggcagt gaggcccaag gagggcaggt 19200 ggggctggac ctgtggctct gctggggagc aggtgggatg tgttaagatg ctgatttcag 19260 ccgggcagct cctccccttc ccttcccagg gagccggggt gtgcagggcc gttccttccc 19320 ctctgggaag tgcagctcct gtgcaccgga gaggcccccg cactgtccct gcccgtccca 19380 ccaactgtcc ctgcccgtcc ctccactgtc cctgcccatc cccctcactg tccctgccca 19440 ttccccgcac tgtccctgct catccccaac actgtccctg cccatccccc cctcaccgtc 19500 cctgcccatc ccccccgcac cgtccctgcc catccccccc cactgtccct gcccaccccc 19560 cactgtccct gcccatcctt gccttgggag gtgctgtcac ctggtggagc catgggaagc 19620 acctctccca cccagggtct ctggttcctg tggcgggtgc agagccagcc acatcactga 19680 caccccagct gggttgggtc cagctgaccc cacaccccca cccctctgct cggcctctgc 19740 ctgtgccccc atccctggcc ctgcatgacc ataaactcct tgagggcagg aagtgagtga 19800 gtcctacccg cactgagtcc ctctcacccc tccttctcat tttggggcgg agggaactgt 19860 cttctgtccc ctgggggtgg cagatggccg cttagcttag aaaggaaaac agcacaattt 19920 agaattcgtc ctgggtgaaa ggtctcggca gcatctgcct cctgctgctt tctggagtcg 19980 catttccatg gagcggccct ggcagggagg gtgggagtgg cacagtggaa atactgaggc 20040 gggaacaaag tggtggcggt agaggtggcg ggagcggcac agcaggcgcc ggggccactt 20100 ggcaccctcc ccccgcagcc ctcctggagt ccccgcccgc cgctcacctg cggggggccc 20160 agataggaca ggttacccca gcgccgccag gccactgagc gtgggggatg ggacaggcag 20220 gtgccgggtg accctaagag ggacgcacgg aaggggagtc cacgggggca cacctggcgt 20280 gggttgggtc tcctccgtgg gagcccgaga gctgggtggc ttggtggaca gggcagctct 20340 ctcgtccagt cgggggagac tggcgttccc tatctcaagc ctcacttttt agccccagag 20400 gggtctcccc accgcccctg ataggaggag atgcggggac ggtggaaggt ggggtgcggg 20460 tcactcatgg aagccctgag gatccccgtg ggcgcggcag ctgttgggtg ccacctcact 20520 cggccaagcc ctggcgccca cctcgcggcc cacacccacc tggccgccgg gacggcattg 20580 aagacagagt ggcccgggta ccgggggtgt ggccagcgcc aaagctgtga aggatttgct 20640 cacaggctgg tggtcctggc catggccatg ctgcaggcct gaggtccctc ccaccttctc 20700 agggacgtct gggtgctgtg cggagggcag gtgttcgccg tctgtagggt gctccagctc 20760 tgtggccatg tgggagagag gtacagactg gggcgggcac aggctggagg ctgcccacag 20820 cagcagcggg agactgggcc caaagctgcc ccccacacac agacccccac acctggctca 20880 aaactttctc caggctcggc ccggttcccc ccagctagcg cgtctcacgg aacctccaag 20940 ccgtggctgt ggggccgtgg caggggcctt ccctgtggtg tccgatgccc aggctgggag 21000 tgggggcgga caatgggccc tctcagcctg cattccccca cgggagcctc ctcaggcctc 21060 cggctggggc tcctgcccca cccgctggga gcagctgccg ccacgtctga cacctgctcc 21120 cagccgccct ttgtccgagc accgacgcct gatttatggg gcgtgttttc ctcttgctgg 21180 cccggcgtga tacagcctcg ttaactggga tgcgtccggt tgggcagatt ctcccacagt 21240 cctccacgtt ggggtccgcg ctcagggtgg ggagtggggc ccggtgtccg ggcagagtgt 21300 cgtggtccat tcagggacag ccaatgtcct gtgacctcag ggcacttggt gggagactgc 21360 tctaggggct cggacctcca gcgtctcccc tctggggtcc cctaaagctg tgggatgggc 21420 ctcccctgcc cctcctcccc gtcttgccca gcaggtgagc ttttcccgct ccttggagcc 21480 tcgaggggcc gggaggcagg ggaccagctt gcatgggcca ggctgaggag cctccagccc 21540 cggctttgat gaccgagcgc cctctgtcct gtacccaccc cctgccgact tcagcggctg 21600 ggaaagcaaa aagggccttt tctccaagca ggagctgtgg ccaactcccg cctgacaatg 21660 gggcatttat gtggagggag ggcgcccctc ctgtggccac agcccagccc agcccagcct 21720 gtgcactcca cgggttccca gcggccgctc cacctcgggg accggccagc ctgtaactgg 21780 atctgcatgc agtgggaacc acgtctgccc tcccaacagg tgcccgcaga ggcaggtctg 21840 tcgctgcagc cccggagtga ccggccacat gggatggagg agggggagcc tcattccggg 21900 gctgtgcccg tcgctcactg ggctggcagg agccgtagca aggcctgagt gaattcccca 21960 gagccccttt ggaaggggtg acatgtgttt tctttaagcc agttggtttg ggacttttgg 22020 cttctggcaa cagatggtcg gccagagtgg cccttttgcg ttctctctac ccaagccgtg 22080 gtcacgggta aaagccttcg gggaagacag atgcctcccg ggcctttggg acaggggcgt 22140 ggctggctgc tgagaaacgc acaggaaccc ccacccccag cctccacaag cccctcgggg 22200 taaggccccc ccaggccact gccacacata gcacaggttt gattggagac actggggtgg 22260 gaataagcgg gagaggaggc gaggacttgg ggggccggtg ggggtcctgt gggaagccgg 22320 cagcttcagg gccggtcctg gggcaggcgg ccgccgcagc cccgcaccca ggtgggctct 22380 ggaaggcggg acggttccct ctgtacatcc catctggagg aggccatgcc cacccagggc 22440 ctgagccgtg aggccgctgc cttcccgggc cgctggggcc gctgaggctg gtggcatgtt 22500 ctgcttcaga ggcttcacca ctgggaagag cggcgcccgg cgggtcccag accttttccc 22560 ggagtgcaag gccaggaggt gactggggcc ggagatttat gaccacagca ggtggctggg 22620 gggggcaggc tctgcgcctg ttgccgcagg gaacaggtga gaccaggcct gggtggagat 22680 gcaggacccc tcccctggct ccccttgttc tctctgagcc cagactgtgt gtccggggtg 22740 aggggcagcc tttcacccac cttgacgggg ttgcagagac ctggcagagg gacctcctcc 22800 tggcaccttg gtttccccgg ctgtaatgtg ggtgcttctg ctcctggctg atgtgcctgg 22860 atgtgagtta tgaagactca atgccgaaag ggaaacgtgt ctgggggctg agcgtgggca 22920 gggaacgggc cagccttcat ccccagtgcg atcttgacat gagatgtccc atccccattg 22980 cagtctcagc ctgaggtgtc ccatcagaaa ggcttcctgg ccgggcgcgg tggcttacgc 23040 ctgtaatccc agcactttgg gaggccgagg cgggcagatc acgaggtcaa gagatcaaga 23100 ccagcctggc caacatggtg aaaccctgtc tctactaaaa atacaaaaat tagctgggca 23160 tggtgtcagg tgcctgtagt cccagctact cgggaggctg aggcaggaga atcgcttgaa 23220 cctgggaggc agaggttgca gtgagcctag atcgcgcccc tgcactccag cctgggcaac 23280 agagcaagac tcaaaaaaaa aaaaaaaaaa aaaagaaaag aaaaagaaag gcttccacgt 23340 aggaaggggc tgatccacac tttccgtggc tgccatcatg ttgacacctg aaggttggaa 23400 aaaccacggc ctttgttact gcccaagcaa tgcctgttcg ctttagcaaa gctgggaaga 23460 aaagcagata ggcctgcaga cccggaggct ctacggagct aatgcacccg gctcctctcc 23520 atggctctcc gtgggtgtag acccagaccc ctgcccgggc tgtggagtcc aagctgggtt 23580 ctgctggaac atcctagtct ctagcttgtt ccccaagtta gcagtcattt gtcttcttct 23640 ttctttcttt ttttgagtct tgctctgtca cccaggctgg agtgcagtgg cgtgatctcg 23700 gctcactgca acctctgcct cctgggttca agtgattctc ctgcctcagc ctcattcagt 23760 ggcattcagt acattcccaa agttgtgcaa tcgcggcatg tgtctggttc cagaacattt 23820 tatcagccct aagggaaccc cgctaagcag ccgttgtgcc cgtgcccggg gtgtgggggg 23880 aggcggctgt gcccatgaca ggttcccggt gctgagtggc gggggcgctg gggaagcagg 23940 tgggaactaa ctgccctggc acatctgcca acagctgtgg cggggccttc gtgggcccgc 24000 gatgccagga ccccaacccg tgcctcagca ccccctgcaa gaacgccggg acatgccacg 24060 tggtggaccg cagaggcgtg gcagactatg cctgcagctg tgccctgggc ttctctgggc 24120 ccctctgcct gacacccctg gacaacgcct gcctcaccaa cccctgccgc aacgggggca 24180 cctgcgacct gctcacgctg acggagtaca agtgccgctg cccgcccggc tggtcaggtg 24240 agggaggacc cacagcctga ggggtgggca gacccaggcc cgccgccagc tgcccaggca 24300 acttgaggta cttggcggga tccaggcccg ggtctctgac ccggaagtaa ttgggagccc 24360 ctgccccagg gtccctgccc ccctcccaga cctggaatcg cctcgttttc agctgcttgc 24420 tctggcccaa ctcttgctgt ccccgcttct gcgagacaag gggagcctct gtgtccaggc 24480 cgcccctgcc cccttcctcc tgccgtgcac tggccctcac ctgttagggt gaagggtgtc 24540 tggaggtgtc agctctggga agagacggcc caggctcagg cctcttggga gctgtggtcc 24600 ttcatctgcc aaaggcgacc ggtctcacgg caggtcctga ggatcaaatg tgatagcacc 24660 taaggtggag cccaggctgg cacctccagg tggcctgaag ggagggaagg cccggcctcg 24720 gggagcagtg aggccagcac gaccctcttg tccccttgtc tccagggaaa tcgtgccagc 24780 aggctgaccc gtgcgcctcc aacccctgcg ccaacggtgg ccagtgcctg cccttcgagg 24840 cctcctacat ctgccactgc ccacccagct tccatggccc cacctgccgg caggatgtca 24900 acgagtgtgg ccagaagccc gggctttgcc gccacggagg cacctgccac aacgaggtcg 24960 gctcctaccg ctgcgtctgc cgcgccaccc acactggccc caactgcgag cggccctacg 25020 tgccctgcag cccctcgccc tgccagaacg ggggcacctg ccgccccacg ggcgacgtca 25080 cccacgagtg tgcctgcctg ccaggtagtg ctgcccgctg gccggggtgc acgagcccct 25140 ccccggctgc caggcccagg gggtgggaac ggggcgcctg gtgtaggggc agccctggga 25200 gggcccatgg cggggagttg ggaaggcggg atggcgagtt ccccagactg cgtgtggtgt 25260 cggggggacg cgtctggagc ggggtgtgcg ctgggaggag gcttgcacag ggtccaggtg 25320 ccagcccgtg tctctggagt ttcccggaac tcacattcga gctgtaggac gccctgcctc 25380 cccccagtgc aggcaccctg acttgcgcaa cccccagggt ttcccctgca gtggggctgg 25440 ggccggagag ggcgtgggag ctgcgggggc accatcactt cccaagccat ggcccttcct 25500 gccatcctgc tggtctcctg cgggtgtggg tgacaggtag gcgcagccgc ctgcccgtgt 25560 ccccagtgct ccccgatccc cgtgttgtac ataggagcct ctctgctgta gttcttgttt 25620 gagtaaattt tgcagcagct ctaaaaataa ggaagagttt tgaaaacctc cagtctcggc 25680 cgcgcgtggt ggctcacgcc tgtactccca gcactttggg aggccgaggt gggcagatca 25740 caaggtcagg agatggagac catcctggct aacacggcga aaccccgtct ctactaaaaa 25800 tacaaaaaat tagccaggca tggtggcacg cgcctgtagt cccagctact caggaggctg 25860 agggaggaga atcacttgaa cccgggaggt ggaggttgca gtgagccgag atcccgccac 25920 tgcactccag cctgggcgac agagcgagac tccgtctcaa aaaaaaaaaa aaaaaagtga 25980 agagaaaaaa gaaaacctcc aatctcagct gcacagaatg gctgttaggt gggaatgggg 26040 agagggcatt caggcagggg tggggctgga gggtgcacag ggtcctggct gacccacggg 26100 tggcccaagg gctgccccac ccccttggaa gccccaagct ggcctggccc ctgcaaaggg 26160 gaagctccct ggctgcccag tcagctgagc ccagccacct ctgcgtgcag acaccagacg 26220 tgtgcgtttc ctgcaagtca ctgggcgagc gtggggtggg gacagacact ccctggccag 26280 cttggatctc tgcagggtgg ggcgcagagc ccttctgctg gtgggggctc agatgccttc 26340 ctagcagcac gggaccctac ctccagtcac acggattgag gggccaagct cttggggggc 26400 cgtggttcag gctagggcag gtggggtgtg tgcggccttg tccaaggcca cctgtgtgca 26460 gagctgatgc ccacccgcct tgggcagggt gggtgaggac ctcctggtga ggcccagcgg 26520 cagagatgcc aggggtgctg ggccggctca gagccactgc ccgtgcaggg gcagctcagg 26580 gccctgggga gtgggcaggg agggaagggg aggggctggt ggggttcact cacttgagtg 26640 tgggtagatc tcagaggctg gggtgctggg ggctgggtca cccgtccggt gaggggtgtg 26700 ctcactgcct gctgtggagt ctggccgttt cagggtgaga tgaccatccg ggggcctccc 26760 caaatgactc aggcctgcca gaccccagct tcagactgtc cccggcggcc cctgcaccca 26820 ggccccctga agggccctca gccacctctg ggtggtcagg agcccaggag acagctgtgt 26880 gtggagtcac ggggaaccga gatgggggcg gccctgagct ctgcaggccc gggaactggc 26940 tgggatttgg ggtcgcacag cctgcctggc acgtcagcac tgtctcctcg tggggtcgct 27000 ggccgaggct acgtcgcaga gggtccagct ggggtccacg gggctctcgc tggcttcccg 27060 ccctctcccc cactgtgtgc ctcagccagt gctttgcagg gcagccctgg ggtcgcaggc 27120 ccagccaggg gccccatctg cctaggagcc ggggtgcagg gccccacaca tgagtgggac 27180 cgggactcag gctccaggct ctgaattttt tttttttttt tgagacaggt ctctgtcact 27240 caggctggag tacagtgacg tgccctcagc tcactgcagc cttgacctcc caggctaaag 27300 cgatccttct gcctcagccc ctcaagtagc ttgggaccac aggccggcgc caccacgctt 27360 tgctacttct tgtatttttt tttgtagaga cggggtttcg ccatgttgtc caggctggtc 27420 ttgaactcct gggcgcaagc gatccaccca cttcagcctc ccaaagtgcc gggattccag 27480 gcgtgagcca ccgcgccagg gctggggtct ggttttgggt gctccccaag ctgatttgca 27540 ttttggattt tggttgtctc tggctgtctg cttttgctcc ggtccagcag acagacagaa 27600 cggacggggt ggtggcggtg gggcagtgtc tcagagggag gggccgagca ggtgggagct 27660 ccaagggcaa cgggggttca aaggcatctc gggagggcgg caattagtaa ccggctgcag 27720 gtttggctgt tgccgaacgc gcctttctgg gttgatcatt gaaccagcgg gagttgagtg 27780 gattaatagc taactggctg ccttggaggc ctgggtgggg ccatacctcg ggggaggggg 27840 ccctggcagc ctgtggagga ggagggggag gaggaggtgg gcaccagagc tctgccccag 27900 ggaggatggg ccatctgaat ctctgcagct tccctggggc tgcgaggggc ccaccctgcc 27960 ccagccagca gcctgctggc cccatgtccc cctgctgctc agagcccagg ccccagtgtc 28020 agcccctgaa gatggggtat gcggcccccc tgtggagaca ttgtagggag ctctgagcat 28080 gcaggcggca cccaggtgca ggagctgccg gaacctctta aaagagaccc aagggttttt 28140 gtgacagaat cttctggaac cctgtgttcc tgagtccctg tttagccagt cctgtccaaa 28200 atgattcttt caagcagggc gtgtccctct gggcactgga gtgaggcagg ggacgggcac 28260 agaccccggg ggccccaggg cagggccgtg gaggccaggc tcttgtgtcc agagcagtgt 28320 gtcgggggtc caggcaggag ccggactgga cctcagggaa gaggctgacc cggcccctct 28380 tgcggcaggc ttcaccggcc agaactgtga ggaaaatatc gacgattgtc caggaaacaa 28440 ctgcaagaac gggggtgcct gtgtggacgg cgtgaacacc tacaactgcc gctgcccgcc 28500 agagtggaca ggcgcgtata cgggtcgccg cagggcgggg tagccggggc ggggctgcta 28560 cccacttact aaactgcact cacaggcata gcggggagca gccgagaccc tgtcccaggc 28620 caggcagact agggttggcc gagaaggaca acatgatggg ggccccactt cccgtggcca 28680 gggtctggtg cccctcacct cgggggtcag cacagacagg gccctggttg gtctgtgtgg 28740 gccgtttgca acctggctcc aggcagcccc tgtgcccagt ggggtgtcct ggcattgagg 28800 gccttcagca ccccactcag gccacgcttc ctgggccagt cacagggatg cctggatgaa 28860 cagacaggcc ctatgttcgg gaattaccct ctggtggggt gacgggcaaa ggtgcacccc 28920 gacgcagcag tgtccacaga gcacaaagag gaggcaagac tccatggtgc tgggcctgca 28980 gaggaaggag tgatttaggc tggcggtggg tggaggagtc agcccaggaa ggcttcctgg 29040 agggggcggc attgccaccc tgcgtcttag gggacaggga gctcagggag tggcagctgc 29100 ccggggccga cagctcctgt tccctgcagg tcagtactgt accgaggatg tggacgagtg 29160 ccagctgatg ccaaatgcct gccagaacgg cgggacctgc cacaacaccc acggtggcta 29220 caactgcgtg tgtgtcaacg gctggactgg tgaggactgc agcgagaaca ttgatgactg 29280 tgccagcgcc gcctgcttcc acggcgccac ctgccatgac cgtgtggcct ccttctactg 29340 cgagtgtccc catggccgca caggtgagtg ccttgaggcc caggtgggcg cagggggctc 29400 acatgggcca ggcctgagct gtgtgacctc acccagggac tgtggccctc ctgcacccag 29460 gataacctga aagggccttt ctggtcaggg aggccggtgg tcgctgtgag tcccccggaa 29520 catcccaagt gtcacgggat gcccgacggg gtcacggcgg gcagctcctc ctggggtgtc 29580 agtggggttg gaccccagcc gtgggtggtg tgccatgcct ggcccagggg ccgtgcccac 29640 tgaccgccgc tgcctgccca ggtctgctgt gccacctcaa cgacgcatgc atcagcaacc 29700 cctgtaacga gggctccaac tgcgacacca accctgtcaa tggcaaggcc atctgcacct 29760 gcccctcggg gtacacgggc ccggcctgca gccaggacgt ggatgagtgc tcgctgggta 29820 ggtgccagca cagggggtgc ggccaggtgg gggtggcagc ctggccccag aaccgatgag 29880 aagtcgattc tggcttcagg gggtgcccag ttagatgggg gatgggaccc tgccagtccg 29940 atgggggtgg tgtgcagtga ggtgttgcag gggccagggt gcctggagca gcctctcacc 30000 cgtgtgccct cgccaggtgc caacccctgc gagcatgcgg gcaagtgcat caacacgctg 30060 ggctccttcg agtgccagtg tctgcagggc tacacgggcc cccgatgcga gatcgacgtc 30120 aacgagtgcg tctcgaaccc gtgccagaac gacgccacct gcctggacca gattggggag 30180 ttccagtgca tctgcatgcc cggtgcgttg gccggggcca gggcgggaaa ccgagtcgag 30240 gctgggcagc cttggagggg cagccccggg aacatctgtg ggttgcttcc gctttcccca 30300 gcctccatgc cttctgggcc cacagcctgc acggggcatg gggaaactga ggccaggcca 30360 cagggccagc agagcacggg ggcagtgcta ggcagatgag ggtgccgggc caggggccac 30420 gggctgcagg ggagcgggtg cgcagggggc ccgtggtggc tgggacactg ggctggaggc 30480 agggttcgtt tctgtcccaa gtccacagct gtgcccagtg gggcattggg gccgcgcgtg 30540 cccccctcac tgttgcccca ccccacaggc tacgagggtg tgcactgcga ggtcaacaca 30600 gacgagtgtg ccagcagccc ctgcctgcac aatggccgct gcctggacaa gatcaatgag 30660 ttccagtgcg agtgccccac gggtgagggc cgcccccgcc ccctgccccc gggtcgtctg 30720 caccctggcc tcctgagggg tgcctgggcg tgggttgtgt cccctgcccc cgggccatct 30780 gcaccccggc ctcctgaggg gtgagggccg cccccacccc ctgccctcgg gccgtccgca 30840 ccccggcctc ctgaggggtg agggccgtgg gttgtgtccc ctgctcctgg gccgtctgca 30900 ccccggcctc ctgagggggg cctggctgtg gattgtgtcc cctgcccctg ggctgtctgc 30960 accccggcct cctgaggggt gcctggccgt aggttgtgtc ccctgcccct gggccgtctg 31020 caccccggcc tcctgagggg ggcctggcca ttggttgtgt tcagattccc aggagagcga 31080 gggttttgcc tcatgagtgg atgggagtgt tttcagactt ccccgaagga agggcagggc 31140 ccagtgggga gtgggagctt gcccaggggt cgcggtggag cccagggcca ggagatcctc 31200 cttgatctgg gtgcagcccc ttctgcctgg agagaagggt ataccaggag ctgcagtgcc 31260 cagacaggga ggaggctcca gcctggcttt ctgagggctc gaggctggga gggaggccag 31320 cctgccctgt cctgccatgt cccctgccca acggcacgcc agcgacaagg gtatgcagag 31380 aaccacgtgg ggcaggttgg cctgcaatgg agatgatggc tgcccggggc ggacttggag 31440 gaactctcag gggcctgggt gaaaggtgtt tccatctgtc ccagagctgg gggccggggc 31500 gcagagccca gggaggcgtt gccagcccaa gcctaggctc ccaagacata gattacccgt 31560 cccagacact ggagcgaggg gagccccatt ctcagcccgg ctccactgta gccatagcaa 31620 cccagtcggt ttgggaaaaa ggccccttct gtgggagcga gggcgtgtgg tgctggggga 31680 ggggctttga tccggggagc gggcagcggg aggcagggag agctggggct ggctgagctc 31740 aggctgagtg tgccctgggg agcccagcca ggcgctcact gctggggtct ggccaggggt 31800 ccctgaaggg ccatagcgct gttgcacatg actccctccc ctcccctccc cggccccagg 31860 cttcactggg catctgtgcc agtacgatgt ggacgagtgt gccagcaccc cctgcaagaa 31920 tggtgccaag tgcctggacg gacccaacac ttacacctgt gtgtgcacgg aaggtgcggg 31980 ccggcgccca ccagcgggga gggactgggg acgggacagg gcactcaggg cagtgaaact 32040 gacaaggtca tggacacctt ggtctgggcc acctggtcca gaggccgaga gcacagcaat 32100 cctgagtagg tgggaatgcc acgctcggag ccctcctcag gttggcacag gtgaccccag 32160 gtctggtcat gggtgtcccg ggggccgcca gtcctaagtc ttcctgtgcc cgcccctccc 32220 gcggtccagg gtacacgggg acgcactgcg aggtggacat cgatgagtgc gaccccgacc 32280 cctgccacta cggctcctgc aaggacggcg tcgccacctt cacctgcctc tgccgcccag 32340 gctacacggg ccaccactgc gagaccaaca tcaacgagtg ctccagccag ccctgccgcc 32400 acgggggcac ctgccaggac cgcgacaacg cctacctctg cttctgcctg aaggggacca 32460 caggtggccg gccaggcggg tggccggcgg ggggccagtg ggcagggcgg gcctgaggac 32520 tgaccgacac gtgccacccc ttcaggaccc aactgcgaga tcaacctgga tgactgtgcc 32580 agcagcccct gcgactcggg cacctgtctg gacaagatcg atggctacga gtgtgcctgt 32640 gagccgggct acacaggtga gcggccctgc acgtgggggc tgactgcact gtgctcagag 32700 gtcaaggtca gccactctgc cctggggctg ctgaggggtt gggtgagggc tttggtgctg 32760 cccacctggg cagaccgtgg tctgcagcag agattgcagc gggaccaggg ctataactgg 32820 cagcacgatg ggtccccccg acccctcttg tctccggtca gagctgtccc aggccgtgtt 32880 ggcagagtgg cccagcaggc caagagggag cagccagccc tgaggccagg cttaagtacc 32940 ttctgcctct gtggctcacc agcacctttg agcaggtccc tctgcctctc tgggcctcag 33000 attctcatca gtcagggctg cagtagcccc tgcccctggg gcccctggga ggatgacttg 33060 agtgggcact ggtgcccggg gaggctggca catggcacat tgtgatccaa gatggtttct 33120 gacacctgga aggatgtggc cagaagggtg atcgccccgg ctcaacagac agggaaatcg 33180 aggttgacgt gggtgggacc ccctgggcgc tgggcctcgg agtctgaccc gccctgccct 33240 tagggagcat gtgtaacatc aacatcgatg agtgtgcggg caacccctgc cacaacgggg 33300 gcacctgcga ggacggcatc aatggcttca cctgccgctg ccccgagggc taccacgacc 33360 ccacctgcct gtctgaggtc aatgagtgca acagcaaccc ctgcgtccac ggggcctgcc 33420 gggacagcct caacgggtat gcggcggggc cgatcatggg gacacatcag tcctaaaccc 33480 tgggagctct gctgccagga gggtggcacc tgcacagagc ttgagatggg ccagaaacgg 33540 gcctcggacg gggctgggtg gcagggagct cccctcgggg acgcacatgc ctctgggtcc 33600 tcagtcagac tacagtccac acccagcagc tgtgtgctgc gcgtccttgt gggctgcatg 33660 gtgtgctcag gacacaggcc cacagtgacc ctggaacacg tttccatggt aacagccgca 33720 agtgtttctg atgcccatga tgtgcctggc agcactctgc ctggatggga ccatttaaat 33780 cagcagggac cccattgccc tcatttggcg cagagaaggc acgtgctttg ttcggggcca 33840 cacagcaggc aagtggtggg gggcttttcg cagcacccta ctgccctgca cttggcatgg 33900 tcccctcgct aatgaaccaa acccctgccg cagtcttggg tatgggaagc gctgcggccc 33960 ccactttttt ctttattttg agacagagcc ttgctctgtt gtccaggctg gagtgcaatg 34020 gcatgatctc agctcactgc aacctccacc cctcaggttc aagtgattcc cctgcctcag 34080 cctcccgagt agctgagatt acaggcatgt gccaccacac ctggctaatt tttgtatttt 34140 tagtatatat ggggtggggg agtttcgcca tgttggccag gctgatcttg aactcctggc 34200 ctcaagtgac tcagccaccc ccgcctccca aagtgctggg attacaggtg tgagccacag 34260 cgcccagcct gcccccacta aaggactctg cgagtctgag tggatgggca cctccgccag 34320 cccatagggc attgcagacc cgggagtgcc caggccccgg ccgtgctgct cgggcctccc 34380 tcgacctgca gtgtggtccc ccttgcaggt acaagtgcga ctgtgaccct gggtggagtg 34440 ggaccaactg tgacatcaac aacaacgagt gtgaatccaa cccttgtgtc aacggcggca 34500 cctgcaaaga catgaccagt ggctacgtgt gcacctgccg ggagggcttc agcggtgagt 34560 gggctgcgcg tttctcagtg cagagggccg ccctcgagtc tggggagctg gagacacagg 34620 atcgggaccc aggtccagcc agcaccatgc atggtgtctc cctcccgtgg caaagcctta 34680 gctccaggcc tctggctctt ggagatgagg aggggcctgg ggtggggagc aggcccaggg 34740 ctgctgttgt ggggccctgc caaggtgcct gggagtgctg gacatgccga gtgctgtccc 34800 cttccctcca ggtcccaact gccagaccaa catcaacgag tgtgcgtcca acccatgtct 34860 gaaccagggc acgtgtattg acgacgttgc cgggtacaag tgcaactgcc tgctgcccta 34920 cacaggtgag gggtgggtgg ggcctatgct ggagggggcg gggcctatgt gggaggggcg 34980 ggacctgtgc tggagagggc ggagcctgtg ctggagaggg tggggccagg ggtgggggcg 35040 gcctgggaag ccgtgactgg agggtaaagt ggtggctttc ttggggcggg gccttccaga 35100 aggttctgcg cctcactgag tgccttaggc ccctgcagta tgtgggcctt ctcccggggg 35160 tggggttggc agattagcaa agatacagga tgcctgggag aaaattttca tgcaagcatg 35220 tgttttacct ggcaagcctg ccgcaagaca caggtaagat gcaggtgaat ccttagcagg 35280 gccgggcctg ggtcactggg ctccgtgctc ccggcgccca ccctgctggg gtctctgggg 35340 gccgggcttc cccacccccg ccagcaggac ctgagggatt cctttgatgg cgggctctct 35400 ttgtgtgagc ggaacaagga gccctctgtt tgggctgagc ggggaggggg atgttccccc 35460 aactgtccag cccctgctat gaacccagcc aaggggatca caggaaatga tctcctctag 35520 aaaagacaaa gaagagtcca caagcaccgg ggtcctggcg ctccccagga gcgggcagag 35580 ggtgctgcca cggggcctgg tcagggaggg cctgcctggg ccaggcagcc gggctggagg 35640 tcccacgcct gcggttccca tgacatccag cagcctgtgg ccagaacgct gttttctttg 35700 accgaagtta aggacctggg ttttctctcc tgtgtcagtc agtgaggacc ctccctccac 35760 ggctgggcgc ccctgtccct ttcagacacg tcgatcgggc cggcaggagg agcggcggtg 35820 ctgacccgct tcctatctgc ccgcttcctg cttcctttcg cacaattgtc ggaaactctg 35880 gcgcagttcc tgcctgcccg ggtcactgtg gaaaccaata ggaagagagg ccctggaaag 35940 tcgccccagc aggccaggga gataatgggg gacagggaag ataacgcagt ccccacggct 36000 ataaacagga agctcggcca tgggcttgtg cagaaaaagg cccttggctg tgcacgtggt 36060 tcctacatag cctttgccag acacgctgcg tggccctgga ggtctccagg ccgtgccccg 36120 tgccccgaca cgcaggatgt gctgggggcc tgggcctgtc ccctccatcg cagcccttgt 36180 ttgttggcct cccgtgatgg gcgtttgctg cctgtgacac ggagccccag gattagtttc 36240 aggcctcggt ctcaggccag ggcctttcct cgtcacccag agatgagggg gcttgcgtcc 36300 ggggtgggcc caggtccctg cagtgtcgtg gagcccaccc gactgaggcc gcctctcctg 36360 gtgcagtcct tgttcctggg cacgcctggt ggggtgaggc ctaggctgtg ccccgctgcc 36420 aaccgggaat gaggggtggc ttctgagcgt gacatttgtg cagcttgtct tcagcgtccc 36480 cccatctgtg ctccactcct ccctgggtca gggtcctggc acagccggga gcgctcaggg 36540 gtctcggtgc acatttgcct cccggcggga ccagcagacg gcactctgat ggcggaaaga 36600 ccagcaggcg gtggccgatt tgggagatcc ctctgggtga ggctcccggg gtcacgtgtg 36660 tctccttccc gcaggtgcca cgtgtgaggt ggtgctggcc ccgtgtgccc ccagcccctg 36720 cagaaacggc ggggagtgca ggcaatccga ggactatgag agcttctcct gtgtctgccc 36780 cacgggctgg caaggtgagg ctggccaggg cccggtgagg gctgggatgg gaggtcagga 36840 tgtctgcggg acacaggcag ctcccaggca ggctagatga gtctttgaag aggagccggt 36900 gggtgctgag gaggccctgg tcagagaagt tctggaatca ggaattgacc tgggagcacc 36960 gttcccaact ccagttcctg tgaccttctt aggccaaaat taggggagag gggatggtcc 37020 tggggtcacg gaagcccact cctgggtcgg gagaggcact gtaggtgggt gggccagcct 37080 gggaagggcc tggagggcca ggggccgctg gtgaccaacc ggcctcctcc tgccccacag 37140 ggcagacctg tgaggtcgac atcaacgagt gcgttctgag cccgtgccgg cacggcgcat 37200 cctgccagaa cacccacggc ggctaccgct gccactgcca ggccggctac agtgggcgca 37260 actgcgagac cgacatcgac gactgccggc ccagtgagta gccccgcggc tctggcctcc 37320 tccaggaagc tctcaggcct cagttccccc gggcagggtt ggtgcgcatg gtgctggcca 37380 taagcaccca gggaggccgg agtgtggccg gggagggttg ggtcaggtgt gggggatgtg 37440 ctgagggatg gccagggtgg ttcaggaggg ccccagagcc ggcttgctcc tccctgcact 37500 tcgcggaaga gtcacgagag cccctcccac ggctcttcac tgagccgagg atggctcggg 37560 cccggcctgc actcccgcct ggctcatcgg gccccgctgc aggccagggc tctcaggcct 37620 cccagccctg cttcacagag gttgaggttg gcagaagccg ggggtcttgc tggcatcacc 37680 tggcaggcag aggcaagaac tgtctctcct cccctgctcg gtctgtgaag cctgcaaagc 37740 tgccccctgc cctggagctt gaggacagga gcaggtgggg agagagaccc caagcacagg 37800 agacgggtgt gacgcagcct gtgggtgctg gggctcccca ccagacacct ttgtcacagg 37860 gctgcttgcc gcagcctcgg caacgaaagg ctgggctgtc cccagccatg cagccttccc 37920 ggccccctcc cgcaggtgtg ggtttgtgcc tgcccctcac actcaccctt ccgtcctctc 37980 ccagacccgt gtcacaacgg gggctcctgc acagacggca tcaacacggc cttctgcgac 38040 tgcctgcccg gcttccgggg cactttctgt gaggaggaca tcaacgagtg tgccagtgac 38100 ccctgccgca acggggccaa ctgcacggac tgcgtggaca gctacacgtg cacctgcccc 38160 gcaggcttca gcgggatcca ctgtgagaac aacacgcctg actgcacaga gaggtgtgcg 38220 gggctcgagt gaggccgtgg aagggaacgg gcggtgcggg ccacacgcag tagctggcag 38280 cctggcacac catgcaggcg tccgcctagg ccggctgggc acttagcgct ggctgcattg 38340 agtccagaca ttgttcattg taccgacctg tccctcctga gagggccatt gtgacctcct 38400 cctgtgtccc gcaccaggag cgccggtagt ctgtcctgaa gaactggtgc gcttccttct 38460 attagacaac gagaactctg tccgttcttg tttccttttt gccgtgtttg ccaggaagct 38520 catttggtaa acgtcagtct cgtccctggt ttctgacgta attacctccc gtgttaccag 38580 gacggtttct ggtttctccc tcatttactt taaccaccct atgcccacgc gtttggtaaa 38640 accactctcg acctgactta taaagaaagc aggggaggga ggtgtgacgt ggtgtgagag 38700 ccgggccccg ggttcccact ggcctccctg ggtcagccag gctgccctgg gcctgtcctg 38760 gccatcaggc ccctagggtt gagcagaagg ggaggtgctg gcgaggcaga atctgctgga 38820 gcggggaccc accaatgccc tccgctcagc ccccgcctgc ccaccccctt gcagctcctg 38880 cttcaacggt ggcacctgcg tggacggcat caactcgttc acctgcctgt gtccacccgg 38940 cttcacgggc agctactgcc agcacgatgt caatgagtgc gactcacagc cctgcctgca 39000 tggcggcacc tgtcaggacg gctgcggctc ctacaggtgc acctgccccc agggctacac 39060 tggccccaac tgccaggtga gtgcgccggc cacagaggtg cccgaaggag gggccctggg 39120 tgggtgcctg cctgcgggag gtgggaacgg tcacccccag gtcccactgt gtcggctcgg 39180 ggtcaccccc acacccccgg gcagagggtt tctggggatc tgagcagccg gtgaaggaac 39240 ctgatgcgga agcagcaggc accttcgttt caatcccagg tttctggagc cggggcagga 39300 gctcaggaat tggggaattg aggaaaagtg ttccttctag caaaggccga gggtggtctg 39360 gacctgctga gggccctgag ggagacccag cccaggctgt tcctggtgtg ggggtggtgg 39420 ggagtccagg ggcggcagtt tccacttctg tagaatgggt tgcagcctgg gttggagtag 39480 gccccttggc agatgtgcgt tctgagctac cggggaaatg gccggggcgc gggcacccag 39540 ctgaccccaa tctgtcccca gaaccttgtg cactggtgtg actcctcgcc ctgcaagaac 39600 ggcggcaaat gctggcagac ccacacccag taccgctgcg agtgccccag cggctggacc 39660 ggcctttact gcgacgtgcc cagcgtgtcc tgtgaggtgg ctgcgcagcg acaaggtaac 39720 ctgctgtgcc cacccggctc gggtcccagc ccatcaaggt cctctgtggg cctgggcctc 39780 acctgtctac caccccatcc cccgcaggtg ttgacgttgc ccgcctgtgc cagcatggag 39840 ggctctgtgt ggacgcgggc aacacgcacc actgccgctg ccaggcgggc tacacaggca 39900 gctactgtga ggacctggtg gacgagtgct cacccagccc ctgccagaac ggggccacct 39960 gcacggacta cctgggcggc tactcctgca aggtgggggt ccctcctagg gtaagggttg 40020 tggccggcac gagtgttgcc acacaccagg ccctggctgg gagctggccc agtggagaaa 40080 actgaacctg ataggcccat gcactgttca gtctcattag gggagggctg gggtaatcag 40140 ggtagactgc ctggaagagg tggcctgtgg aaagcctgaa ggagggtacc tatactgaga 40200 agtgggatgg ggttttccct tcccctagat tgtgtctggg cttggccaac acctaccctg 40260 aggccctcac ctctatccta tgggacgggg tccacccacc cccaacaggc agtgactccg 40320 gtcaccgagg ccccgccagg gtctgttggg ctgggtctct ctccaggtct gacaggagcg 40380 aggggcccgt ggcttcgctg gacctgaggg cagctcatgt ggccctgtca gccctcacag 40440 tggggtgtgg gagcactgca tcctggcgcc ggctgagccg aagggcccct cgttctgtcg 40500 cctgcacagt gcgtggccgg ctaccacggg gtgaactgct ctgaggagat cgacgagtgc 40560 ctctcccacc cctgccagaa cgggggcacc tgcctcgacc tccccaacac ctacaagtgc 40620 tcctgcccac ggggcactca gggtaagggc cgctgcacgg agggctggtg ttggccatcc 40680 atggccaggg caggggcagg gcaggcaccc cgggaccggc cagaggcatc caggaaccag 40740 ccagaaactc ccattttcct gtgtgaggcc aaggccgact cacagctgcc cagggaaagg 40800 gcccagagcg ggggtcccag tcggaagggc gtctccacgg cacccttgac acctgcctct 40860 cccgagtgtc cgtgcagccc cagacctgag cgcttgtctt ccgggacgga cacgcggcac 40920 ggcagggccg gggtgtggcg ggcttgggcc actgacgaaa cctggccccg caggtgtgca 40980 ctgtgagatc aacgtggacg actgcaatcc ccccgttgac cccgtgtccc ggagccccaa 41040 gtgctttaac aacggcacct gcgtggacca ggtgggcggc tacagctgca cctgcccgcc 41100 gggcttcgtg ggtgagcgct gtgaggggga tgtcaacgag tgcctgtcca atccctgcga 41160 cgcccgtggc acccagaact gcgtgcagcg cgtcaatgac ttccactgcg agtgccgtgc 41220 tggtcacacc ggtgggtgcc gcgcccaggc gggtggggcg tgtggggcag cagggtgagc 41280 ctctcactgc cctgctctta cccctagggc gccgctgcga gtccgtcatc aatggctgca 41340 aaggcaagcc ctgcaagaat gggggcacct gcgccgtggc ctccaacacc gcccgcgggt 41400 tcatctgcaa gtgccctgcg gtaggtgcag gggtgcaggg aggcaggggc ccgccagggg 41460 agacacctgg agaggtccac gtgggggcct cgggacgcag accgggcagt gatcctcccg 41520 gccttcatcc tcctcctcac cctgatgtct tttttttttt tttagtttca ataaatgatt 41580 ttagagacat tttagattta tagaaaaatg gagcaggaag tagactccct ggggtggctg 41640 tcccctgcac gcagttcccc tggttagcag cttgcatcaa tttcactctg gatttcagtg 41700 atacattgtt accaacagca gcccttcctg tacctggggc ttgcccttgg ctttgtggtt 41760 gtgggtttgg gctgaggtat gacatgcctg cccaccctcg caggatcacg gcagagagtc 41820 cctgccctaa agtcctcagc actccaccag tttacccctt cctccatctc ccgaccccct 41880 ggcacccccg atctttctct gttggtagaa tgtgtgtaaa gggttttgct gctgggggca 41940 aggttgcagg ccgcctccca ggttagagga gagcggtggc actgctggcc gagggctggg 42000 tgtgaggtgg cgggggggcg gggggtggcc caccccgaca ccgtcctgtc ttccctctcg 42060 ggcagggctt cgagggcgcc acgtgtgaga atgacgctcg tacctgcggc agcctgcgct 42120 gcctcaacgg cggcacatgc atctccggcc cgcgcagccc cacctgcctg tgcctgggcc 42180 ccttcacggg ccccgaatgc cagttcccgg ccagcagccc ctgcctgggc ggcaacccct 42240 gctacaacca ggggacctgt gagcccacat ccgagagccc cttctaccgt tgcctgtgcc 42300 ccgccaaatt caacgggctc ttgtgccaca tcctggacta cagcttcggg ggtggggccg 42360 ggcgcgacat ccccccgccg ctgatcgagg aggcgtgcga gctgcccgag tgccaggagg 42420 acgcgggcaa caaggtctgc agcctgcagt gcaacaacca cgcgtgcggc tgggacggcg 42480 gtgactgctc cctcaacttc aatgacccct ggaagaactg cacgcagtct ctgcagtgct 42540 ggaagtactt cagtgacggc cactgtgaca gccagtgcaa ctcagccggc tgcctcttcg 42600 acggctttga ctgccagcgt gcggaaggcc agtgcaagta aggctgcggg gctcatgggg 42660 ctgagggagg acctgaactt ggatgtggcc tggcttgggc ccggaggcca gcatgcagtt 42720 ctaaggctct gctcaggggg tgcagggacg tcccccgcgg ctggccagtg ggctggaggc 42780 accggacggc gggtgcgagg ccccccgagg aaggcggcct gagcgtgtcc cgccccccac 42840 agccccctgt acgaccagta ctgcaaggac cacttcagcg acgggcactg cgaccagggc 42900 tgcaacagcg cggagtgcga gtgggacggg ctggactgtg cggagcatgt acccgagagg 42960 ctggcggccg gcacgctggt ggtggtggtg ctgatgccgc cggagcagct gcgcaacagc 43020 tccttccact tcctgcggga gctcagccgc gtgctgcaca ccaacgtggt cttcaagcgt 43080 gacgcacacg gccagcagat gatcttcccc tactacggcc gcgaggagga gctgcgcaag 43140 caccccatca agcgtgccgc cgagggctgg gccgcacctg acgccctgct gggccaggtg 43200 aaggcctcgc tgctccctgg tggcagcgag ggtgggcggc ggcggaggga gctggacccc 43260 atggacgtcc gcgggtgagt gagacccggc gcccacggtc aatccccgca actctcctgg 43320 gccctccccg acggcctccc tgcccctcac ggccggcgcc atggcaagca gtactctccc 43380 cactttatgg taaaagagac ggaggttccg agaggacctg ggacttcagg gcctgcgcag 43440 gcaaggagta gaggcagcat tccagctcag ggccctgacc ccacagccac acccttttcc 43500 tgggccactg ccttcccctg gacaggcggc actcctgtgc ccagtaggtg attttgagat 43560 tgagctgtgc cttaggcact ggatactaca ctgattaaaa ctcagccctc tgccaggcga 43620 ggaggctcac acctgtaatc ccagcacttt gggaggctga ggcaggcgga gcccttgagc 43680 ccaggagttc gagaccagtc tgggcaacat agggagacct tgtctctgtt tttttaaaaa 43740 aagtattaaa agaagtaaaa aacaaaacac tcagctctcc aggggttccc acagggctga 43800 acagccccac cccagacaag aatgccgctt ggtcatggcg tctgcctggc ttgggctgga 43860 gaggaggcag gtggaggtcc tgggaggggg cattgctggg ccttgcagtt ggaggaggag 43920 ctggtggggg tggggggtcc cacggtggga gaacacaggc aggagcagct tgagtgcagg 43980 gggcacccca caaggctccc gcccatgcct actcgatctg gggcagctgg acccaggagc 44040 caggttggtt gtgcccttcg tgtgcctgac cctggtgggt ttgcctgtca gttctgctgg 44100 cttggagcaa tcctggaggt cagaagcatc atctcacagg ctggatggga tcctgctcac 44160 gggaacccag tcctggggag cagagctcac ccccagccag cctcaccaca cagcccacca 44220 ccgctgcacc cacccccacc tcacgcctgt gcttcctgca gggcctgggg atggctcctg 44280 ggggaggact gggctcctgg cacatactct gtcctgagat gaggaaacgt gtctgtggca 44340 ccagcaggag ccagagaggg tgtcagggcg ggccagggag agcgcgttgg tgggtatctg 44400 ggatgagccg tgatcagcac tggccggagt cggggggctg gcaccagtcc cctgcagggt 44460 agctgctgtc agacctggct tcccaccacc ccaggctgcc tcaccatgtc ctgactgtgg 44520 cgtcatgggc ctcagtgtcc tgcggcagca tccctggccg gtgggcgggg gaggaggaag 44580 cctcgggtcc cagcccctct ctgattgtcc gcccagctcc atcgtctacc tggagattga 44640 caaccggcag tgtgtgcagg cctcctcgca gtgcttccag agtgccaccg atgtggccgc 44700 attcctggga gcgctcgcct cgctgggcag cctcaacatc ccctacaaga tcgaggccgt 44760 gcagagtaag tgtggcccca tcccgggaac aggctctgcc tgcagggggt gccatccccc 44820 cgtgccccag acacgctggc tgtttgtgcc agttgctacc cacgggtgtg agcgttgccg 44880 tccgagttgg ggtagggctt ttctggaatt ttctgaatgg cactccgccc ccacctgcgg 44940 cggtcacagc tgccggtgga gccacctggg aacgagtcca gccacgggaa agtgggtgcc 45000 tgcttctctc cccacccttt cctcctgaat tttctttgtt gggtattatt tcaaaatcat 45060 tacggctttt tttaaagaaa aaaaaagaga gagagagaag aattgatcgg tgtcatgtga 45120 agtgttgaag tttgtatctt gaaaatccct ctaaatcctt tgtcttaaca gctcagtgcg 45180 agtgcagcga tttgaagttg actaatcctc cttccttaaa ggagaaaaaa gtaaaagccg 45240 tctccagata gagtcggctg gtgcaggaga gaatttagcg atagtttgca attctgatta 45300 atcgcgtaga aaatgacctt attttggagg gcgggatgga ggagagtggg tgaggaggcg 45360 cccggacgcg gagccagtcc gccgcccccc ggccaccagc ctgctgcgta gccgctgcct 45420 gatgtccggg cacctgcccc tggcccccgt gcccgcaggt gagaccgtgg agccgccccc 45480 gccggcgcag ctgcacttca tgtacgtggc ggcggccgcc tttgtgcttc tgttcttcgt 45540 gggctgcggg gtgctgctgt cccgcaagcg ccggcggcag catggccagc tctggttccc 45600 tgagggcttc aaagtgtctg aggccagcaa gaagaagcgg cgggagcccc tcggcgagga 45660 ctccgtgggc ctcaagtgag cggacgccgc ccctgcttct gggtccccag tgggaggcca 45720 ggcccgggcc aggggtctct gggggcttcc tagggagctc gctcagcctc acttctcgac 45780 ccctcacccc ccaggcccct gaagaacgct tcagacggtg ccctcatgga cgacaaccag 45840 aatgagtggg gggacgagga cctggagacc aagaagttcc gggtgagtcg cgaggctccc 45900 gggctcctgg gctcccgggc acctgccgcc gggctgccct gacaggctct gctcactccc 45960 tctatgtagt tcgaggagcc cgtggttctg cctgacctgg acgaccagac agaccaccgg 46020 cagtggactc agcagcacct ggatgccgct gacctgcgca tgtctgccat ggcccccaca 46080 ccgccccagg gtgaggttga cgccgactgc atggacgtca atgtccgcgg gcctggtaag 46140 ggtgccagca gccagggctt ccctagcccc gtggcccacc tgcctctctc ccctaagccc 46200 cgaggctggg gtacagttga tactctggaa acttagaatt gggggtgaga gctttcatcc 46260 ttggggtgtt tcccattcag agtagacgtg ggggggtcct ggagtcctcc tgttcttcca 46320 ccaacccctt cctggggtga taccgcaggg ccactctgtc cctgtaaatt actcttttct 46380 gaaaccttct tgagacatgg aaagcgttga ttttcttttt cttttttttt tttctttttt 46440 tttgtttttg agatggagtc tcgctccgtc acccaggctg gagtgcagtg gcacgatctc 46500 ggctccctgc aacctccacc tctcgggttc aagcaattct cctgcctcag cctccccagt 46560 agctgggact acaggcgcct gccaccacac ctggctaatt tttgtatttt tagcagcgat 46620 ggggtttcac catgttggcc aggctggtct cgaactcctg acctcaggtg atccgcccac 46680 ctcggcctcc cccagtgcta ggatgacagc gtgagcctcc gcatcctgct gaagcattaa 46740 ttttctaact gcatgcttct ggggtcctct ttttcctggg tggattttgg gtgccaggtc 46800 ttgggcaggt caggaagcag ttgcccgcca ggagtttaag ctggattcgg ctctgtccac 46860 taagcagccc aggcggactt cagctgcccc ttggtggctg tgtgcacggg gccaccaagt 46920 gctgggtggt gcatccacac cgtggcccct tgagcttggg gctgctgccc ccctccccct 46980 gggctgcagc tggggaccgg ccctccagac tgagcacccg tctctgcctc tgcagatggc 47040 ttcaccccgc tcatgatcgc ctcctgcagc gggggcggcc tggagacggg caacagcgag 47100 gaagaggagg acgcgccggc cgtcatctcc gacttcatct accagggcgc cagcctgcac 47160 aaccagacag accgcacggg cgagaccgcc ttgcacctgg ccgcccgcta ctcacgctct 47220 gatgccgcca agcgcctgct ggaggccagc gcagatgcca acatccagga caacatgggc 47280 cgcaccccgc tgcatgcggc tgtgtctgcc gacgcacaag gtgtcttcca ggtaggcagt 47340 ggctgcctgt gtgcccacct gccctcctca gggccgcctg gtggtctggg gcagtggcca 47400 ggcttacgtg gccctgggag cctgaccccg agcacagctg agtccgggac aactggtgcc 47460 tccacctggg accttcgcag tcagcgaggt gcgaggggga gggcgtcggg cccatctgtg 47520 ttctccaggg aagtcaggca gaggcgggtc tggcaggagg cctgggggat ctgctgagtg 47580 aggcagcacc tccccacccc cagcaaaaca ggctccatca gggttgtggg ccttgctcaa 47640 ggtccaggtt ccactgctgc agcccctcgc agccccgccc ctccctcaac cttggctgcc 47700 ggcgtagcct gtggcagtga gaagcagggt ttagaggctg ccgctcggtg cctgcagacc 47760 tcgggctcag cttgccggtg agctcgtggc aagaatggat ttagggattt ggatgcctgg 47820 gtctccaggg agtgtccctg gcaggggctg cctttgcagt cacccctgct gcgagtcccc 47880 aggctccagg cggccctgga gcaagcaggt tcagatgggc acagcccggg gagttaccac 47940 agagcttctc atttcctgat ttcttagctc aggtgacaca ttgtcatctc gcagaaataa 48000 atgtaggtga gcagaaagag cgtggaaggg agccccgtgc gggtttggtg tgtgcctctc 48060 tcagctgcct tctttgcaca gatgtggaag ttcccaggtg ctttgcagga atcaggccaa 48120 gttgcctttt gcacccgcca gtgagcaggg gccattcctc ctgccagatg ccaagacccg 48180 gctgcattac aagggctgcc caccccctct ctgggcagag ccggacacta gctcggcggt 48240 tctgagacgg ctgtagggcc gtgagcccgg cttcctgagt gccagctgta accgccgttg 48300 ggggcaggga cttgacctct ctgttttgta ggtggtcatg gcagctagga ccacagtgag 48360 gattaaatga ggccacacgt ggtcacgatg gatcccactg tcaccaaggg gcctgtctgt 48420 gggcagcaca gcccctgccc ccatccgggc ccctcctcag gggcagcccc atggcgtttc 48480 gtcttgcccg gccgtgccga tctcaggagg gtctcgtctg tgtccggaag acagtggggc 48540 ttcccgtcca ggcgttcgtt ctgggcagga ggcatcggtg tacgtctgcc cagcacccgc 48600 ctgagcctct ccctgttgcc cagatcctga tccggaaccg agccacagac ctggatgccc 48660 gcatgcatga tggcacgacg ccactgatcc tggctgcccg cctggccgtg gagggcatgc 48720 tggaggacct catcaactca cacgccgacg tcaacgccgt agatgacctg ggtgagccca 48780 cgggggcacg gctgctctgt cgtggggcgg gaccgccaca gggacgggtg ggactgggtt 48840 gcctccagct ggtctccaac ctaccccatc tgcttctttc acgcaggcaa gtccgccctg 48900 cactgggccg ccgccgtgaa caatgtggat gccgcagttg tgctcctgaa gaacggggct 48960 aacaaagata tgcagaacaa cagggtgagc gcgaggctgg gatgccaggg gagacgtgag 49020 ggctgaatcc acagagaagt gagggccaaa cccacggggc gcagggacac aagggcctga 49080 cccacagggt ctcgagggtc gctgggcccg catgtgggcc ccacccgcat gatgcgggca 49140 cctcgttagt gctcctgccc tccttcagcc cctgtttatg gagcccttcc aagtgcaggt 49200 ccagctgtca ggacacggcg gctcgcccct cagacccagc cctaccctcc tgggcggcag 49260 tcatggcagg gcacacagcc tggcgtgggg aacgctgctg ctgccactgc tgtgtcaccg 49320 tcacctggac ctcaccttct gtccccagct gtcaccaggc gggggtgggg atgggaatgt 49380 ggtgacgcag gtggtgcaga ttgagccaga acacgcgtgg cggcagctcc ctgcggggcg 49440 gggcctcttg gtgtgttcac caaggccaag gacctcaagg ctcagaggaa gaagtctcag 49500 gactatatcc aaggggagcc acccccagcc ctcatcctgg ccctgcagct cctggccctg 49560 cagctgctgt tggtttcccc tgggtgctca gggcacaggt gcagacaccc ccacctccct 49620 gccgccagaa cccccacccc cgcccccaac tcctgctgcc cccttggcat gtcaggctca 49680 ggcgtctctc cctcctgggt gagggcacac agcgggcctg ggcaccgggg catgttggcc 49740 caggcgctcc ccagtccgtg gcagcatggc cccacagaga ctgggcccca gagggcatca 49800 aggcctggga agccccttcc cactcccaca cagcagcctc acccagacct gtgacgtgtc 49860 caccgcacag aggagacccc aggaaggagc ttggtggcca ccagggtggc ttgatggccg 49920 tccagatgac gagctccctc cctggtgccc tcgccggtgc ctctgaaccg ctccaggaat 49980 ttccttttgt gccttattgg gggcagggaa gagggcctgc agttggttag attttcagtg 50040 gggtctgtga cccccccata gaggtggagc cccgctgatc tagggtagag gactgcacag 50100 atcccctctc tgggtgggtt tcagaagatg tatcaaagcc ttaacattta acaagagtca 50160 ggctaggtgg ttgcaggacg ctggggtggg gtcctgagga gcagcctgcc tgcccccacc 50220 ccgcggagga ggttgtactg ctgcttcctc tggtgatgga accttgggga gggtccccac 50280 gcctggcctg gcccccctca ccggcccccg ccctcatccc ccaggaggag acacccctgt 50340 ttctggccgc ccgggagggc agctacgaga ccgccaaggt gctgctggac cactttgcca 50400 accgggacat cacggatcat atggaccgcc tgccgcgcga catcgcacag gagcgcatgc 50460 atcacgacat cgtgaggctg ctggacgagt acaacctggt gcgcagcccg cagctgcacg 50520 gagccccgct ggggggcacg cccaccctgt cgcccccgct ctgctcgccc aacggctacc 50580 tgggcagcct caagcccggc gtgcagggca agaaggtccg caagcccagc agcaaaggcc 50640 tggcctgtgg aagcaaggag gccaaggacc tcaaggcacg gaggaagaag tcccaggatg 50700 gcaagggctg cctgctggac agctccggca tgctctcgcc cgtggactcc ctggagtcac 50760 cccatggcta cctgtcagac gtggcctcgc cgccactgct gccctccccg ttccagcagt 50820 ctccgtccgt gcccctcaac cacctgcctg ggatgcccga cacccacctg ggcatcgggc 50880 acctgaacgt ggcggccaag cccgagatgg cggcgctggg tgggggcggc cggctggcct 50940 ttgagactgg cccacctcgt ctctcccacc tgcctgtggc ctctggcacc agcaccgtcc 51000 tgggctccag cagcggaggg gccctgaatt tcactgtggg cgggtccacc agtttgaatg 51060 gtcaatgcga gtggctgtcc cggctgcaga gcggcatggt gccgaaccaa tacaaccctc 51120 tgcgggggag tgtggcacca ggccccctga gcacacaggc cccctccctg cagcatggca 51180 tggtaggccc gctgcacagt agccttgctg ccagcgccct gtcccagatg atgagctacc 51240 agggcctgcc cagcacccgg ctggccaccc agcctcacct ggtgcagacc cagcaggtgc 51300 agccacaaaa cttacagatg cagcagcaga acctgcagcc agcaaacatc cagcagcagc 51360 aaagcctgca gccgccacca ccaccaccac agccgcacct tggcgtgagc tcagcagcca 51420 gcggccacct gggccggagc ttcctgagtg gagagccgag ccaggcagac gtgcagccac 51480 tgggccccag cagcctggcg gtgcacacta ttctgcccca ggagagcccc gccctgccca 51540 cgtcgctgcc atcctcgctg gtcccacccg tgaccgcagc ccagttcctg acgcccccct 51600 cgcagcacag ctactcctcg cctgtggaca acacccccag ccaccagcta caggtgcctg 51660 agcacccctt cctcaccccg tcccctgagt cccctgacca gtggtccagc tcgtccccgc 51720 attccaacgt ctccgactgg tccgagggcg tctccagccc tcccaccagc atgcagtccc 51780 agatcgcccg cattccggag gccttcaagt aaacggcgcg ccccacgaga ccccggcttc 51840 ctttcccaag ccttcgggcg tctgtgtgcg ctctgtggat gccagggccg accagaggag 51900 cctttttaaa acacatgttt ttatacaaaa taagaacaag gattttaatt ttttttagta 51960 tttatttatg tacttttatt ttacacagaa acactgcctt tttatttata tgtactgttt 52020 tatctggccc caggtagaaa cttttatcta ttctgagaaa acaagcaagt tctgagagcc 52080 agggttttcc tacgtaggat gaaaagattc ttctgtgttt ataaaatata aacaaagatt 52140 catgatttat aaatgccatt tatttattga ttcctttttt caaaatccaa aaagaaatga 52200 tgttggagaa gggaagttga acgagcatag tccaaaaagc tcctggggcg tccaggccgc 52260 gccctttccc cgacgcccac ccaaccccaa gccagcccgg ccgctccacc agcatcacct 52320 gcctgttagg agaagctgca tccagaggca aacggaggca aagctggctc accttccgca 52380 cgcggattaa tttgcatctg aaataggaaa caagtgaaag catatgggtt agatgttgcc 52440 atgtgtttta gatggtttct tgcaagcatg cttgtgaaaa tgtgttctcg gagtgtgtat 52500 gccaagagtg cacccatggt accaatcatg aatctttgtt tcaggttcag tattatgtag 52560 ttgttcgttg gttatacaag ttcttggtcc ctccagaacc accccggccc cctgcccgtt 52620 cttgaaatgt aggcatcatg catgtcaaac atgagatgtg tggactgtgg cacttgcctg 52680 ggtcacacac ggaggcatcc tacccttttc tggggaaaga cactgcctgg gctgaccccg 52740 gtggcggccc cagcacctca gcctgcacag tgtcccccag gttccgaaga agatgctcca 52800 gcaacacagc ctgggcccca gctcgcggga cccgaccccc cgtgggctcc cgtgttttgt 52860 aggagacttg ccagagccgg gcacattgag ctgtgcaacg ccatgggctg cgtcctttgg 52920 tcctgtcccc gcagccctgg cagggggcat gcggtcgggc aggggctgga gggaggcggg 52980 ggctgccctt gggccacccc tcctagtttg ggaggagcag atttttgcaa taccaagtat 53040 agcctatggc agaaaaaatg tctgtaaata tgtttttaaa ggtggatttt gtttaaaaaa 53100 tcttaatgaa tgagtctgtt gtgtgtcatg ccagtgaggg acgtcagact cggctcagct 53160 cggggagcct tagccgccca tgcactgggg acgctccgct gccgtgccgc ctgcactcct 53220 cagggcagcc tcccccggct ctacgggggc cgcgtggtgc catccccagg gggcatgacc 53280 agatgcgtcc caagatgttg atttttactg tgttttataa aatagagtgt agtttacaga 53340 aaaagacttt aaaagtgatc tacatgagga actgtagatg atgtattttt ttcatctttt 53400 ttgttaactg atttgcaata aaaatgatac tgatggtgat ctggcttcca ctcccctctg 53460 ctctggcctt tggctccctt tctgggaggg aggcagggct gctatgctct gagggagcca 53520 ggagtcgagg gccccttctg ctgggagagt gacggtgagg ctgcctagtc ctggcccacg 53580 ggggtgtggg gaccacgctg cctccaggga ctccatggtg tctgcagcct gcctggtcca 53640 ggccctttgt agggagatgg acacacagca gcaagggggt tgcagcccta tgggaggtgg 53700 ggctcgtgcc tggggtgaca cggctcccag gacaccatgc gtgagtctgg ccctcctggc 53760 agctcggggc tcttctcctc tcagcctcgg agggatgaag gtcccaccca gccatcctgg 53820 ggacagcccc tcagggagct ctgcagcagg cagggggctg catagggagg gccttgaggc 53880 agtggcatga cccctctacg ggctggagac cacagggagg actctggccc ctataggagg 53940 gcaaagggag ctgtcgaagc ctatgagcag ggcagcatgg gtcgtggcag agtctggaaa 54000 gttgtgtgat cctaaggaac tggctgagcg aggcagaggc ggcctggtcc tggggccctg 54060 cccctgaata gagctgccag cctcacacaa gggtgggccc cttctctccc cactgcctgg 54120 gcctctgccc agccccagac cttcagggca ggccagtggc ttcaaaccag agcggtgggg 54180 agtctgagat ccctctttgg attgcaaagc actgcctgcc ctgggcccag tctctccaag 54240 gagggatgtg agcccgaggc ctctactatg gctgggggct gcgtctgcca gccagcgctg 54300 ggcaccagga ccaggagggg ccaccgtgga actgcagtga gtggcctgac tcttgtcttc 54360 aaagggggtg acccagccgg agtcctgccc ataaaactcc cagcaccctg aaattccact 54420 cctgggggtc tgtccgaaag aagtgaaaac agggactcaa acaaacgcac gtggccactt 54480 gctgtcccag catcactcag tacagccaca gacagcctga gcgtccactg ccaacgacgg 54540 gtcagcaaaa ccgtctgctg tgacggtgaa ccttagtgtc agcttggccg ggccatttcg 54600 ccaaacggag tgtgtctctg aggtgttctg gatgaggttc gcatttgcat ccacagcccg 54660 ggtaaggcag tccgccctcc ccagtgtggg tgggccccgt gcggtccgct gaggcccggg 54720 gagaacaata ggccgagtgc caggggtcct cccacccaac cgcttgcact ggacattggt 54780 cttttcctgc cttcagactt ggactgaaaa cgtgggctct tcctgggcct ggagcccacc 54840 ggccttcgga ctcgcaacgc catcaggcct cctgcctgca actgcagacc ctgggaactg 54900 caggcctcga ggtcgtgtgg ccaattccct gcaataacct ctccagtggc acgtcttatg 54960 ggctccgctt gccggaagaa ccctgacgaa tgcgcacgag g 55001 5 19 DNA Artificial Sequence PCR Primer 5 cgggtccacc agtttgaat 19 6 20 DNA Artificial Sequence PCR Primer 6 ttgtattggt tcggcaccat 20 7 17 DNA Artificial Sequence PCR Probe 7 tcccggctgc agagcgg 17 8 19 DNA Artificial Sequence PCR Primer 8 gaaggtgaag gtcggagtc 19 9 20 DNA Artificial Sequence PCR Primer 9 gaagatggtg atgggatttc 20 10 20 DNA Artificial Sequence PCR Probe 10 caagcttccc gttctcagcc 20 11 7693 DNA H. sapiens CDS (1)...(7671) misc_feature 2672, 2673 n = A,T,C or G 11 atg ccg ccg ctc ctg gcg ccc ctg ctc tgc ctg gcg ctg ctg ccc gcg 48 Met Pro Pro Leu Leu Ala Pro Leu Leu Cys Leu Ala Leu Leu Pro Ala 1 5 10 15 ctc gcc gca cga ggc ccg cga tgc tcc cag ccc ggt gag acc tgc ctg 96 Leu Ala Ala Arg Gly Pro Arg Cys Ser Gln Pro Gly Glu Thr Cys Leu 20 25 30 aat ggc ggg aag tgt gaa gcg gcc aat ggc acg gag gcc tgc gtc tgt 144 Asn Gly Gly Lys Cys Glu Ala Ala Asn Gly Thr Glu Ala Cys Val Cys 35 40 45 ggc ggg gcc ttc gtg ggc ccg cga tgc cag gac ccc aac ccg tgc ctc 192 Gly Gly Ala Phe Val Gly Pro Arg Cys Gln Asp Pro Asn Pro Cys Leu 50 55 60 agc acc ccc tgc aag aac gcc ggg aca tgc cac gtg gtg gac cgc aga 240 Ser Thr Pro Cys Lys Asn Ala Gly Thr Cys His Val Val Asp Arg Arg 65 70 75 80 ggc gtg gca gac tat gcc tgc agc tgt gcc ctg ggc ttc tct ggg ccc 288 Gly Val Ala Asp Tyr Ala Cys Ser Cys Ala Leu Gly Phe Ser Gly Pro 85 90 95 ctc tgc ctg aca ccc ctg gac aac gcc tgc ctc acc aac ccc tgc cgc 336 Leu Cys Leu Thr Pro Leu Asp Asn Ala Cys Leu Thr Asn Pro Cys Arg 100 105 110 aac ggg ggc acc tgc gac ctg ctc acg ctg acg gag tac aag tgc cgc 384 Asn Gly Gly Thr Cys Asp Leu Leu Thr Leu Thr Glu Tyr Lys Cys Arg 115 120 125 tgc ccg ccc ggc tgg tca ggg aaa tcg tgc cag cag gct gac ccg tgc 432 Cys Pro Pro Gly Trp Ser Gly Lys Ser Cys Gln Gln Ala Asp Pro Cys 130 135 140 gcc tcc aac ccc tgc gcc aac ggt ggc cag tgc ctg ccc ttc gag gcc 480 Ala Ser Asn Pro Cys Ala Asn Gly Gly Gln Cys Leu Pro Phe Glu Ala 145 150 155 160 tcc tac atc tgc cac tgc cca ccc agc ttc cat ggc ccc acc tgc cgg 528 Ser Tyr Ile Cys His Cys Pro Pro Ser Phe His Gly Pro Thr Cys Arg 165 170 175 cag gat gtc aac gag tgt ggc cag aag ccc agg ctt tgc cgc cac gga 576 Gln Asp Val Asn Glu Cys Gly Gln Lys Pro Arg Leu Cys Arg His Gly 180 185 190 ggc acc tgc cac aac gag gtc ggc tcc tac cgc tgc gtc tgc cgc gcc 624 Gly Thr Cys His Asn Glu Val Gly Ser Tyr Arg Cys Val Cys Arg Ala 195 200 205 acc cac act ggc ccc aac tgc gag cgg ccc tac gtg ccc tgc agc ccc 672 Thr His Thr Gly Pro Asn Cys Glu Arg Pro Tyr Val Pro Cys Ser Pro 210 215 220 tcg ccc tgc cag aac ggg ggc acc tgc cgc ccc acg ggc gac gtc acc 720 Ser Pro Cys Gln Asn Gly Gly Thr Cys Arg Pro Thr Gly Asp Val Thr 225 230 235 240 cac gag tgt gcc tgc ctg cca ggc ttc acc ggc cag aac tgt gag gaa 768 His Glu Cys Ala Cys Leu Pro Gly Phe Thr Gly Gln Asn Cys Glu Glu 245 250 255 aat atc gac gat tgt cca gga aac aac tgc aag aac ggg ggt gcc tgt 816 Asn Ile Asp Asp Cys Pro Gly Asn Asn Cys Lys Asn Gly Gly Ala Cys 260 265 270 gtg gac ggc gtg aac acc tac aac tgc ccg tgc ccg cca gag tgg aca 864 Val Asp Gly Val Asn Thr Tyr Asn Cys Pro Cys Pro Pro Glu Trp Thr 275 280 285 ggt cag tac tgt acc gag gat gtg gac gag tgc cag ctg atg cca aat 912 Gly Gln Tyr Cys Thr Glu Asp Val Asp Glu Cys Gln Leu Met Pro Asn 290 295 300 gcc tgc cag aac ggc ggg acc tgc cac aac acc cac ggt ggc tac aac 960 Ala Cys Gln Asn Gly Gly Thr Cys His Asn Thr His Gly Gly Tyr Asn 305 310 315 320 tgc gtg tgt gtc aac ggc tgg act ggt gag gac tgc agc gag aac att 1008 Cys Val Cys Val Asn Gly Trp Thr Gly Glu Asp Cys Ser Glu Asn Ile 325 330 335 gat gac tgt gcc agc gcc gcc tgc ttc cac ggc gcc acc tgc cat gac 1056 Asp Asp Cys Ala Ser Ala Ala Cys Phe His Gly Ala Thr Cys His Asp 340 345 350 cgt gtg gcc tcc ttt tac tgc gag tgt ccc cat ggc cgc aca ggt ctg 1104 Arg Val Ala Ser Phe Tyr Cys Glu Cys Pro His Gly Arg Thr Gly Leu 355 360 365 ctg tgc cac ctc aac gac gca tgc atc agc aac ccc tgt aac gag ggc 1152 Leu Cys His Leu Asn Asp Ala Cys Ile Ser Asn Pro Cys Asn Glu Gly 370 375 380 tcc aac tgc gac acc aac cct gtc aat ggc aag gcc atc tgc acc tgc 1200 Ser Asn Cys Asp Thr Asn Pro Val Asn Gly Lys Ala Ile Cys Thr Cys 385 390 395 400 ccc tcg ggg tac acg ggc ccg gcc tgc agc cag gac gtg gat gag tgc 1248 Pro Ser Gly Tyr Thr Gly Pro Ala Cys Ser Gln Asp Val Asp Glu Cys 405 410 415 tcg ctg ggt gcc aac ccc tgc gag cat gcg ggc aag tgc atc aac acg 1296 Ser Leu Gly Ala Asn Pro Cys Glu His Ala Gly Lys Cys Ile Asn Thr 420 425 430 ctg ggc tcc ttc gag tgc cag tgt ctg cag ggc tac acg ggc ccc cga 1344 Leu Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly Tyr Thr Gly Pro Arg 435 440 445 tgc gag atc gac gtc aac gag tgc gtc tcg aac ccg tgc cag aac gac 1392 Cys Glu Ile Asp Val Asn Glu Cys Val Ser Asn Pro Cys Gln Asn Asp 450 455 460 gcc acc tgc ctg gac cag att ggg gag ttc cag tgc atg tgc atg ccc 1440 Ala Thr Cys Leu Asp Gln Ile Gly Glu Phe Gln Cys Met Cys Met Pro 465 470 475 480 ggc tac gag ggt gtg cac tgc gag gtc aac aca gac gag tgt gcc agc 1488 Gly Tyr Glu Gly Val His Cys Glu Val Asn Thr Asp Glu Cys Ala Ser 485 490 495 agc ccc tgc ctg cac aat ggc cgc tgc ctg gac aag atc aat gag ttc 1536 Ser Pro Cys Leu His Asn Gly Arg Cys Leu Asp Lys Ile Asn Glu Phe 500 505 510 cag tgc gag tgc ccc acg ggc ttc act ggg cat ctg tgc cag tac gat 1584 Gln Cys Glu Cys Pro Thr Gly Phe Thr Gly His Leu Cys Gln Tyr Asp 515 520 525 gtg gac gag tgt gcc agc acc ccc tgc aag aat ggt gcc aag tgc ctg 1632 Val Asp Glu Cys Ala Ser Thr Pro Cys Lys Asn Gly Ala Lys Cys Leu 530 535 540 gac gga ccc aac act tac acc tgt gtg tgc acg gaa ggg tac acg ggg 1680 Asp Gly Pro Asn Thr Tyr Thr Cys Val Cys Thr Glu Gly Tyr Thr Gly 545 550 555 560 acg cac tgc gag gtg gac atc gat gag tgc gac ccc gac ccc tgc cac 1728 Thr His Cys Glu Val Asp Ile Asp Glu Cys Asp Pro Asp Pro Cys His 565 570 575 tac ggc tcc tgc aag gac ggc gtc gcc acc ttc acc tgc ctc tgc cgc 1776 Tyr Gly Ser Cys Lys Asp Gly Val Ala Thr Phe Thr Cys Leu Cys Arg 580 585 590 cca ggc tac acg ggc cac cac tgc gag acc aac atc aac gag tgc tcc 1824 Pro Gly Tyr Thr Gly His His Cys Glu Thr Asn Ile Asn Glu Cys Ser 595 600 605 agc cag ccc tgc cgc cta cgg ggc acc tgc cag gac ccg gac aac gcc 1872 Ser Gln Pro Cys Arg Leu Arg Gly Thr Cys Gln Asp Pro Asp Asn Ala 610 615 620 tac ctc tgc ttc tgc ctg aag ggg acc aca gga ccc aac tgc gag atc 1920 Tyr Leu Cys Phe Cys Leu Lys Gly Thr Thr Gly Pro Asn Cys Glu Ile 625 630 635 640 aac ctg gat gac tgt gcc agc agc ccc tgc gac tcg ggc acc tgt ctg 1968 Asn Leu Asp Asp Cys Ala Ser Ser Pro Cys Asp Ser Gly Thr Cys Leu 645 650 655 gac aag atc gat ggc tac gag tgt gcc tgt gag ccg ggc tac aca ggg 2016 Asp Lys Ile Asp Gly Tyr Glu Cys Ala Cys Glu Pro Gly Tyr Thr Gly 660 665 670 agc atg tgt aac agc aac atc gat gag tgt gcg ggc aac ccc tgc cac 2064 Ser Met Cys Asn Ser Asn Ile Asp Glu Cys Ala Gly Asn Pro Cys His 675 680 685 aac ggg ggc acc tgc gag gac ggc atc aat ggc ttc acc tgc cgc tgc 2112 Asn Gly Gly Thr Cys Glu Asp Gly Ile Asn Gly Phe Thr Cys Arg Cys 690 695 700 ccc gag ggc tac cac gac ccc acc tgc ctg tct gag gtc aat gag tgc 2160 Pro Glu Gly Tyr His Asp Pro Thr Cys Leu Ser Glu Val Asn Glu Cys 705 710 715 720 aac agc aac ccc tgc gtc cac ggg gcc tgc cgg gac agc ctc aac ggg 2208 Asn Ser Asn Pro Cys Val His Gly Ala Cys Arg Asp Ser Leu Asn Gly 725 730 735 tac aag tgc gac tgt gac cct ggg tgg agt ggg acc aac tgt gac atc 2256 Tyr Lys Cys Asp Cys Asp Pro Gly Trp Ser Gly Thr Asn Cys Asp Ile 740 745 750 aac aac aac gag tgt gaa tcc aac cct tgt gtc aac ggc ggc acc tgc 2304 Asn Asn Asn Glu Cys Glu Ser Asn Pro Cys Val Asn Gly Gly Thr Cys 755 760 765 aaa gac atg acc agt ggc atc gtg tgc acc tgc cgg gag ggc ttc agc 2352 Lys Asp Met Thr Ser Gly Ile Val Cys Thr Cys Arg Glu Gly Phe Ser 770 775 780 ggt ccc aac tgc cag acc aac atc aac gag tgt gcg tcc aac cca tgt 2400 Gly Pro Asn Cys Gln Thr Asn Ile Asn Glu Cys Ala Ser Asn Pro Cys 785 790 795 800 ctg aac aag ggc acg tgt att gac gac gtt gcc ggg tac aag tgc aac 2448 Leu Asn Lys Gly Thr Cys Ile Asp Asp Val Ala Gly Tyr Lys Cys Asn 805 810 815 tgc ctg ctg ccc tac aca ggt gcc acg tgt gag gtg gtg ctg gcc ccg 2496 Cys Leu Leu Pro Tyr Thr Gly Ala Thr Cys Glu Val Val Leu Ala Pro 820 825 830 tgt gcc ccc agc ccc tgc aga aac ggc ggg gag tgc agg caa tcc gag 2544 Cys Ala Pro Ser Pro Cys Arg Asn Gly Gly Glu Cys Arg Gln Ser Glu 835 840 845 gac tat gag agc ttc tcc tgt gtc tgc ccc acg gct ggg gcc aaa ggg 2592 Asp Tyr Glu Ser Phe Ser Cys Val Cys Pro Thr Ala Gly Ala Lys Gly 850 855 860 cag acc tgt gag gtc gac atc aac gag tgc gtt ctg agc ccg tgc cgg 2640 Gln Thr Cys Glu Val Asp Ile Asn Glu Cys Val Leu Ser Pro Cys Arg 865 870 875 880 cac ggc gca tcc tgc cag aac acc cac ggc gnn tac cgc tgc cac tgc 2688 His Gly Ala Ser Cys Gln Asn Thr His Gly Xaa Tyr Arg Cys His Cys 885 890 895 cag gcc ggc tac agt ggg cgc aac tgc gag acc gac atc gac gac tgc 2736 Gln Ala Gly Tyr Ser Gly Arg Asn Cys Glu Thr Asp Ile Asp Asp Cys 900 905 910 cgg ccc aac ccg tgt cac aac ggg ggc tcc tgc aca gac ggc atc aac 2784 Arg Pro Asn Pro Cys His Asn Gly Gly Ser Cys Thr Asp Gly Ile Asn 915 920 925 acg gcc ttc tgc gac tgc ctg ccc ggc ttc cgg ggc act ttc tgt gag 2832 Thr Ala Phe Cys Asp Cys Leu Pro Gly Phe Arg Gly Thr Phe Cys Glu 930 935 940 gag gac atc aac gag tgt gcc agt gac ccc tgc cgc aac ggg gcc aac 2880 Glu Asp Ile Asn Glu Cys Ala Ser Asp Pro Cys Arg Asn Gly Ala Asn 945 950 955 960 tgc acg gac tgc gtg gac agc tac acg tgc acc tgc ccc gca ggc ttc 2928 Cys Thr Asp Cys Val Asp Ser Tyr Thr Cys Thr Cys Pro Ala Gly Phe 965 970 975 agc ggg atc cac tgt gag aac aac acg cct gac tgc aca gag agc tcc 2976 Ser Gly Ile His Cys Glu Asn Asn Thr Pro Asp Cys Thr Glu Ser Ser 980 985 990 tgc ttc aac ggt ggc acc tgc gtg gac ggc atc aac tcg ttc acc tgc 3024 Cys Phe Asn Gly Gly Thr Cys Val Asp Gly Ile Asn Ser Phe Thr Cys 995 1000 1005 ctg tgt cca ccc ggc ttc acg ggc agc tac tgc cag cac gta gtc aat 3072 Leu Cys Pro Pro Gly Phe Thr Gly Ser Tyr Cys Gln His Val Val Asn 1010 1015 1020 gag tgc gac tca cga ccc tgc ctg cta ggc ggc acc tgt cag gac ggt 3120 Glu Cys Asp Ser Arg Pro Cys Leu Leu Gly Gly Thr Cys Gln Asp Gly 1025 1030 1035 1040 cgc ggt ctc cac agg tgc acc tgc ccc cag ggc tac act ggc ccc aac 3168 Arg Gly Leu His Arg Cys Thr Cys Pro Gln Gly Tyr Thr Gly Pro Asn 1045 1050 1055 tgc cag aac ctt gtg cac tgg tgt gac tcc tcg ccc tgc aag aac ggc 3216 Cys Gln Asn Leu Val His Trp Cys Asp Ser Ser Pro Cys Lys Asn Gly 1060 1065 1070 ggc aaa tgc tgg cag acc cac acc cag tac cgc tgc gag tgc ccc agc 3264 Gly Lys Cys Trp Gln Thr His Thr Gln Tyr Arg Cys Glu Cys Pro Ser 1075 1080 1085 ggc tgg acc ggc ctt tac tgc gac gtg ccc agc gtg tcc tgt gag gtg 3312 Gly Trp Thr Gly Leu Tyr Cys Asp Val Pro Ser Val Ser Cys Glu Val 1090 1095 1100 gct gcg cag cga caa ggt gtt gac gtt gcc cgc ctg tgc cag cat gga 3360 Ala Ala Gln Arg Gln Gly Val Asp Val Ala Arg Leu Cys Gln His Gly 1105 1110 1115 1120 ggg ctc tgt gtg gac gcg ggc aac acg cac cac tgc cgc tgc cag gcg 3408 Gly Leu Cys Val Asp Ala Gly Asn Thr His His Cys Arg Cys Gln Ala 1125 1130 1135 ggc tac aca ggc agc tac tgt gag gac ctg gtg gac gag tgc tca ccc 3456 Gly Tyr Thr Gly Ser Tyr Cys Glu Asp Leu Val Asp Glu Cys Ser Pro 1140 1145 1150 agc ccc tgc cag aac ggg gcc acc tgc acg gac tac ctg ggc ggc tac 3504 Ser Pro Cys Gln Asn Gly Ala Thr Cys Thr Asp Tyr Leu Gly Gly Tyr 1155 1160 1165 tcc tgc aag tgc gtg gcc ggc tac cac ggg gtg aac tgc tct gag gag 3552 Ser Cys Lys Cys Val Ala Gly Tyr His Gly Val Asn Cys Ser Glu Glu 1170 1175 1180 atc gac gag tgc ctc tcc cac ccc tgc cag aac ggg ggc acc tgc ctc 3600 Ile Asp Glu Cys Leu Ser His Pro Cys Gln Asn Gly Gly Thr Cys Leu 1185 1190 1195 1200 gac ctc ccc aac acc tac aag tgc tcc tgc cca cgg ggc act cag ggt 3648 Asp Leu Pro Asn Thr Tyr Lys Cys Ser Cys Pro Arg Gly Thr Gln Gly 1205 1210 1215 gtg cac tgt gag atc aac gtg gac gac tgc aat ccc ccc gtt gac ccc 3696 Val His Cys Glu Ile Asn Val Asp Asp Cys Asn Pro Pro Val Asp Pro 1220 1225 1230 gtg tcc cgg agc ccc aag tgc ttt aac aac ggc acc tgc gtg gac cag 3744 Val Ser Arg Ser Pro Lys Cys Phe Asn Asn Gly Thr Cys Val Asp Gln 1235 1240 1245 gtg ggc ggc tac agc tgc acc tgc ccg ccg ggc ttc gtg ggt gag cgc 3792 Val Gly Gly Tyr Ser Cys Thr Cys Pro Pro Gly Phe Val Gly Glu Arg 1250 1255 1260 tgt gag ggg gat gtc aac gag tgc ctg tcc aat ccc tgc gac gcc cgt 3840 Cys Glu Gly Asp Val Asn Glu Cys Leu Ser Asn Pro Cys Asp Ala Arg 1265 1270 1275 1280 ggc acc cag aac tgc gtg cag cgc gtc aat gac ttc cac tgc gag tgc 3888 Gly Thr Gln Asn Cys Val Gln Arg Val Asn Asp Phe His Cys Glu Cys 1285 1290 1295 cgt gct ggt cac acc ggg cgc cgc tgc gag tcc gtc atc aat ggc tgc 3936 Arg Ala Gly His Thr Gly Arg Arg Cys Glu Ser Val Ile Asn Gly Cys 1300 1305 1310 aaa ggc aag ccc tgc aag aat ggg ggc acc tgc gcc gtg gcc tcc aac 3984 Lys Gly Lys Pro Cys Lys Asn Gly Gly Thr Cys Ala Val Ala Ser Asn 1315 1320 1325 acc gcc cgc ggg ttc atc tgc aag tgc cct gcg ggc ttc gag ggc gcc 4032 Thr Ala Arg Gly Phe Ile Cys Lys Cys Pro Ala Gly Phe Glu Gly Ala 1330 1335 1340 acg tgt gag aat gac gct cgt acc tgc ggc agc ctg cgc tgc ctc aac 4080 Thr Cys Glu Asn Asp Ala Arg Thr Cys Gly Ser Leu Arg Cys Leu Asn 1345 1350 1355 1360 ggc ggc aca tgc atc tcc ggc ccg cgc agc ccc acc tgc ctg tgc ctg 4128 Gly Gly Thr Cys Ile Ser Gly Pro Arg Ser Pro Thr Cys Leu Cys Leu 1365 1370 1375 ggc ccc ttc acg ggc ccc gaa tgc cag ttc ccg gcc agc agc ccc tgc 4176 Gly Pro Phe Thr Gly Pro Glu Cys Gln Phe Pro Ala Ser Ser Pro Cys 1380 1385 1390 ctg ggc ggc aac ccc tgc tac aac cag ggg acc tgt gag ccc aca tcc 4224 Leu Gly Gly Asn Pro Cys Tyr Asn Gln Gly Thr Cys Glu Pro Thr Ser 1395 1400 1405 gag agc ccc ttc tac cgt tgc ctg tgc ccc gcc aaa ttc aac ggg ctc 4272 Glu Ser Pro Phe Tyr Arg Cys Leu Cys Pro Ala Lys Phe Asn Gly Leu 1410 1415 1420 ttg tgc cac atc ctg gac tac agc ttc ggg ggt ggg gcc ggg cgc gac 4320 Leu Cys His Ile Leu Asp Tyr Ser Phe Gly Gly Gly Ala Gly Arg Asp 1425 1430 1435 1440 atc ccc ccg ccg ctg atc gag gag gcg tgc gag ctg ccc gag tgc cag 4368 Ile Pro Pro Pro Leu Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln 1445 1450 1455 gag gac gcg ggc aac aag gtc tgc agc ctg cag tgc aac aac cac gcg 4416 Glu Asp Ala Gly Asn Lys Val Cys Ser Leu Gln Cys Asn Asn His Ala 1460 1465 1470 tgc ggc tgg gac ggc ggt gac tgc tcc ctc aac ttc aat gac ccc tgg 4464 Cys Gly Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp 1475 1480 1485 aag aac tgc acg cag tct ctg cag tgc tgg aag tac ttc agt gac ggc 4512 Lys Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly 1490 1495 1500 cac tgt gac agc cag tgc aac tca gcc ggc tgc ctc ttc gac ggc ttt 4560 His Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe 1505 1510 1515 1520 gac tgc cag cgt gcg gaa ggc cag tgc aac ccc ctg tac gac cag tac 4608 Asp Cys Gln Arg Ala Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr 1525 1530 1535 tgc aag gac cac ttc agc gac ggg cac tgc gac cag ggc tgc aac agc 4656 Cys Lys Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser 1540 1545 1550 gcg gag tgc gag tgg gac ggg ctg gac tgt gcg gag cat gta ccc gag 4704 Ala Glu Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu 1555 1560 1565 agg ctg gcg gcc ggc acg ctg gtg gtg gtg gtg ctg atg ccg ccg gag 4752 Arg Leu Ala Ala Gly Thr Leu Val Val Val Val Leu Met Pro Pro Glu 1570 1575 1580 cag ctg cgc aac agc tcc ttc cac ttc ctg cgg gag ctc agc cgc gtg 4800 Gln Leu Arg Asn Ser Ser Phe His Phe Leu Arg Glu Leu Ser Arg Val 1585 1590 1595 1600 ctg cac acc aac gtg gtc ttc aag cgt gac gca cac ggc cag cag atg 4848 Leu His Thr Asn Val Val Phe Lys Arg Asp Ala His Gly Gln Gln Met 1605 1610 1615 atc ttc ccc tac tac ggc cgc gag gag gag ctg cgc aag cac ccc atc 4896 Ile Phe Pro Tyr Tyr Gly Arg Glu Glu Glu Leu Arg Lys His Pro Ile 1620 1625 1630 aag cgt gcc gcc gag ggc tgg gcc gca cct gac gcc ctg ctg ggc cag 4944 Lys Arg Ala Ala Glu Gly Trp Ala Ala Pro Asp Ala Leu Leu Gly Gln 1635 1640 1645 gtg aag gcc tcg ctg ctc cct ggt ggc agc gag ggt ggg cgg cgg cgg 4992 Val Lys Ala Ser Leu Leu Pro Gly Gly Ser Glu Gly Gly Arg Arg Arg 1650 1655 1660 agg gag ctg gac ccc atg gac gtc cgc ggc tcc atc gtc tac ctg gag 5040 Arg Glu Leu Asp Pro Met Asp Val Arg Gly Ser Ile Val Tyr Leu Glu 1665 1670 1675 1680 att gac aac cgg cag tgt gtg cag gcc tcc tcg cag tgc ttc cag agt 5088 Ile Asp Asn Arg Gln Cys Val Gln Ala Ser Ser Gln Cys Phe Gln Ser 1685 1690 1695 gcc acc gac gtg gcc gca ttc ctg gga gcg ctc gcc tcg ctg ggc agc 5136 Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser Leu Gly Ser 1700 1705 1710 ctc aac atc ccc tac aag atc gag gcc gtg cag agt gag acc gtg gag 5184 Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Gln Ser Glu Thr Val Glu 1715 1720 1725 ccg ccc ccg ccg gcg cag ctg cac ttc atg tac gtg gcg gcg gcc gcc 5232 Pro Pro Pro Pro Ala Gln Leu His Phe Met Tyr Val Ala Ala Ala Ala 1730 1735 1740 ttt gtg ctt ctg ttc ttc gtg ggc tgc ggg gtg ctg ctg tcc cgc aag 5280 Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser Arg Lys 1745 1750 1755 1760 cgc cgg cgg cag cat ggc cag ctc tgg ttc cct gag ggc ttc aaa gtg 5328 Arg Arg Arg Gln His Gly Gln Leu Trp Phe Pro Glu Gly Phe Lys Val 1765 1770 1775 tct gag gcc agc aag aag aag cgg cgg gag ccc ctc ggc gag gac tcc 5376 Ser Glu Ala Ser Lys Lys Lys Arg Arg Glu Pro Leu Gly Glu Asp Ser 1780 1785 1790 gtg ggc ctc aag ccc ctg aag aac gct tca gac ggt gcc ctc atg gac 5424 Val Gly Leu Lys Pro Leu Lys Asn Ala Ser Asp Gly Ala Leu Met Asp 1795 1800 1805 gac aac cag aat gag tgg ggg gac gag gac ctg gag acc aag aag ttc 5472 Asp Asn Gln Asn Glu Trp Gly Asp Glu Asp Leu Glu Thr Lys Lys Phe 1810 1815 1820 cgg ttc gag gag ccc gtg gtt ctg cct gac ctg gac gac cag aca gac 5520 Arg Phe Glu Glu Pro Val Val Leu Pro Asp Leu Asp Asp Gln Thr Asp 1825 1830 1835 1840 cac cgg cag tgg act cag cag cac ctg gat gcc gct gac ctg cgc atg 5568 His Arg Gln Trp Thr Gln Gln His Leu Asp Ala Ala Asp Leu Arg Met 1845 1850 1855 tct gcc atg gcc ccc aca ccg ccc cag ggt gag gtt gac gcc gac tgc 5616 Ser Ala Met Ala Pro Thr Pro Pro Gln Gly Glu Val Asp Ala Asp Cys 1860 1865 1870 atg gac gtc aat gtc cgc ggg cct gat ggc ttc acc ccg ctc atg atc 5664 Met Asp Val Asn Val Arg Gly Pro Asp Gly Phe Thr Pro Leu Met Ile 1875 1880 1885 gcc tcc tgc agc ggg ggc ggc ctg gag acg ggc aac agc gag gaa gag 5712 Ala Ser Cys Ser Gly Gly Gly Leu Glu Thr Gly Asn Ser Glu Glu Glu 1890 1895 1900 gag gac gcg ccg gcc gtc atc tcc gac ttc atc tac cag ggc gcc agc 5760 Glu Asp Ala Pro Ala Val Ile Ser Asp Phe Ile Tyr Gln Gly Ala Ser 1905 1910 1915 1920 ctg cac aac cag aca gac cgc acg ggc gag acc gcc ttg cac ctg gcc 5808 Leu His Asn Gln Thr Asp Arg Thr Gly Glu Thr Ala Leu His Leu Ala 1925 1930 1935 gcc cgc tac tca cgc tct gat gcc gcc aag cgc ctg ctg gag gcc agc 5856 Ala Arg Tyr Ser Arg Ser Asp Ala Ala Lys Arg Leu Leu Glu Ala Ser 1940 1945 1950 gca gat gcc aac atc cag gac aac atg ggc cgc acc ccg ctg cat gcg 5904 Ala Asp Ala Asn Ile Gln Asp Asn Met Gly Arg Thr Pro Leu His Ala 1955 1960 1965 gct gtg tct gcc gac gca caa ggt gtc ttc cag atc ctg atc cgg aac 5952 Ala Val Ser Ala Asp Ala Gln Gly Val Phe Gln Ile Leu Ile Arg Asn 1970 1975 1980 cga gcc aca gac ctg gat gcc cgc atg cat gat ggc acg acg cca ctg 6000 Arg Ala Thr Asp Leu Asp Ala Arg Met His Asp Gly Thr Thr Pro Leu 1985 1990 1995 2000 atc ctg gct gcc cgc ctg gcc gtg gag ggc atg ctg gag gac ctc atc 6048 Ile Leu Ala Ala Arg Leu Ala Val Glu Gly Met Leu Glu Asp Leu Ile 2005 2010 2015 aac tca cac gcc gac gtc aac gcc gta gat gac ctg ggc aag tcc gcc 6096 Asn Ser His Ala Asp Val Asn Ala Val Asp Asp Leu Gly Lys Ser Ala 2020 2025 2030 ctg cac tgg gcc gcc gcc gtg aac aat gtg gat gcc gca gtt gtg ctc 6144 Leu His Trp Ala Ala Ala Val Asn Asn Val Asp Ala Ala Val Val Leu 2035 2040 2045 ctg aag aac ggg gct aac aaa gat atg cag aac aac agg gag gag aca 6192 Leu Lys Asn Gly Ala Asn Lys Asp Met Gln Asn Asn Arg Glu Glu Thr 2050 2055 2060 ccc ctg ttt ctg gcc gcc cgg gag ggc agc tac gag acc gcc aag gtg 6240 Pro Leu Phe Leu Ala Ala Arg Glu Gly Ser Tyr Glu Thr Ala Lys Val 2065 2070 2075 2080 ctg ctg gac cac ttt gcc aac cgg gac atc acg gat cat atg gac cgc 6288 Leu Leu Asp His Phe Ala Asn Arg Asp Ile Thr Asp His Met Asp Arg 2085 2090 2095 ctg ccg cgc gac atc gca cag gag cgc atg cat cac gac atc gtg agg 6336 Leu Pro Arg Asp Ile Ala Gln Glu Arg Met His His Asp Ile Val Arg 2100 2105 2110 ctg ctg gac gag tac aac ctg gtg cgc agc ccg cag ctg cac gga gcc 6384 Leu Leu Asp Glu Tyr Asn Leu Val Arg Ser Pro Gln Leu His Gly Ala 2115 2120 2125 ccg ctg ggg ggc acg ccc acc ctg tcg ccc ccg ctc tgc tcg ccc aac 6432 Pro Leu Gly Gly Thr Pro Thr Leu Ser Pro Pro Leu Cys Ser Pro Asn 2130 2135 2140 ggc tac ctg ggc agc ctc aag ccc ggc gtg cag ggc aag aag gtc cgc 6480 Gly Tyr Leu Gly Ser Leu Lys Pro Gly Val Gln Gly Lys Lys Val Arg 2145 2150 2155 2160 aag ccc agc agc aaa ggc ctg gcc tgt gga agc aag gag gcc aag gac 6528 Lys Pro Ser Ser Lys Gly Leu Ala Cys Gly Ser Lys Glu Ala Lys Asp 2165 2170 2175 ctc aag gca cgg agg aag aag tcc cag gat ggc aag ggc tgc ctg ctg 6576 Leu Lys Ala Arg Arg Lys Lys Ser Gln Asp Gly Lys Gly Cys Leu Leu 2180 2185 2190 gac agc tcc ggc atg ctc tcg ccc gtg gac tcc ctg gag tca ccc cat 6624 Asp Ser Ser Gly Met Leu Ser Pro Val Asp Ser Leu Glu Ser Pro His 2195 2200 2205 ggc tac ctg tca gac gtg gcc tcg ccg cca ctg ctg ccc tcc ccg ttc 6672 Gly Tyr Leu Ser Asp Val Ala Ser Pro Pro Leu Leu Pro Ser Pro Phe 2210 2215 2220 cag cag tct ccg tcc gtg ccc ctc aac cac ctg cct ggg atg ccc gac 6720 Gln Gln Ser Pro Ser Val Pro Leu Asn His Leu Pro Gly Met Pro Asp 2225 2230 2235 2240 acc cac ctg ggc atc ggg cac ctg aac gtg gcg gcc aag ccc gag atg 6768 Thr His Leu Gly Ile Gly His Leu Asn Val Ala Ala Lys Pro Glu Met 2245 2250 2255 gcg gcg ctg ggt ggg ggc ggc cgg ctg gcc ttt gag act ggc cca cct 6816 Ala Ala Leu Gly Gly Gly Gly Arg Leu Ala Phe Glu Thr Gly Pro Pro 2260 2265 2270 cgt ctc tcc cac ctg cct gtg gcc tct ggc acc agc acc gtc ctg ggc 6864 Arg Leu Ser His Leu Pro Val Ala Ser Gly Thr Ser Thr Val Leu Gly 2275 2280 2285 tcc agc agc gga ggg gcc ctg aat ttc act gtg ggc ggg tcc acc agt 6912 Ser Ser Ser Gly Gly Ala Leu Asn Phe Thr Val Gly Gly Ser Thr Ser 2290 2295 2300 ttg aat ggt caa tgc gag tgg ctg tcc cgg ctg cag agc ggc atg gtg 6960 Leu Asn Gly Gln Cys Glu Trp Leu Ser Arg Leu Gln Ser Gly Met Val 2305 2310 2315 2320 ccg aac caa tac aac cct ctg cgg ggg agt gtg gca cca ggc ccc ctg 7008 Pro Asn Gln Tyr Asn Pro Leu Arg Gly Ser Val Ala Pro Gly Pro Leu 2325 2330 2335 agc aca cag gcc ccc tcc ctg cag cat ggc atg gta ggc ccg ctg cac 7056 Ser Thr Gln Ala Pro Ser Leu Gln His Gly Met Val Gly Pro Leu His 2340 2345 2350 agt agc ctt gct gcc agc gcc ctg tcc cag atg atg agc tac cag ggc 7104 Ser Ser Leu Ala Ala Ser Ala Leu Ser Gln Met Met Ser Tyr Gln Gly 2355 2360 2365 ctg ccc agc acc cgg ctg gcc acc cag cct cac ctg gtg cag acc cag 7152 Leu Pro Ser Thr Arg Leu Ala Thr Gln Pro His Leu Val Gln Thr Gln 2370 2375 2380 cag gtg cag cca caa aac tta cag atg cag cag cag aac ctg cag cca 7200 Gln Val Gln Pro Gln Asn Leu Gln Met Gln Gln Gln Asn Leu Gln Pro 2385 2390 2395 2400 gca aac atc cag cag cag caa agc ctg cag ccg cca cca cca cca cca 7248 Ala Asn Ile Gln Gln Gln Gln Ser Leu Gln Pro Pro Pro Pro Pro Pro 2405 2410 2415 cag ccg cac ctt ggc gtg agc tca gca gcc agc ggc cac ctg ggc cgg 7296 Gln Pro His Leu Gly Val Ser Ser Ala Ala Ser Gly His Leu Gly Arg 2420 2425 2430 agc ttc ctg agt gga gag ccg agc cag gca gac gtg cag cca ctg ggc 7344 Ser Phe Leu Ser Gly Glu Pro Ser Gln Ala Asp Val Gln Pro Leu Gly 2435 2440 2445 ccc agc agc ctg gcg gtg cac act att ctg ccc cag gag agc ccc gcc 7392 Pro Ser Ser Leu Ala Val His Thr Ile Leu Pro Gln Glu Ser Pro Ala 2450 2455 2460 ctg ccc acg tcg ctg cca tcc tcg ctg gtc cca ccc gtg acc gca gcc 7440 Leu Pro Thr Ser Leu Pro Ser Ser Leu Val Pro Pro Val Thr Ala Ala 2465 2470 2475 2480 cag ttc ctg acg ccc ccc tcg cag cac agc tac tcc tcg cct gtg gac 7488 Gln Phe Leu Thr Pro Pro Ser Gln His Ser Tyr Ser Ser Pro Val Asp 2485 2490 2495 aac acc ccc agc cac cag cta cag gtg cct gag cac ccc ttc ctg acc 7536 cct tcg ccg gag tcg ccc gac caa tgg tcg tcc tcg tcg ccg cac tct 7584 Pro Ser Pro Glu Ser Pro Asp Gln Trp Ser Ser Ser Ser Pro His Ser 2500 2505 2510 aat gtg tct gac tgg tct gag ggc gtg tcg tcg ccc ccg acc tcc atg 7632 Asn Val Ser Asp Trp Ser Glu Gly Val Ser Ser Pro Pro Thr Ser Met 2515 2520 2525 cag tcc cag atc gcg cgc atc ccg gag gcg ttc aag taa tagctcgagg 7681 Gln Ser Gln Ile Ala Arg Ile Pro Glu Ala Phe Lys 2530 2535 2540 tgccagcagc tc 7693 12 423 DNA Homo sapiens CDS (374)...(423) misc_feature 149, 150 n = A,T,C or G 12 agagagagag agaagaattg atcggtgtca tgtgaagtgt tgaagtttgt atcttgaaaa 60 tccctctaaa tcctttgtct taacagctca gtgcgagtgc agcgatttga agttgactat 120 ccctccgtcc ttaaaggaga aaaaagtann agccgtctcc agatagagtc ggctggtgca 180 ggagagaatt tagcgatagt ttgcaattct gattaatcgc gtagaaaatg accttatttt 240 ggagggcggg atggaggaga gtgggtgagg aggcgcccgg acgcggagcc agtccgccgc 300 cccccggcca ccagcctgct gcgtagccgc tgcctgatgt ccgggcacct gcccctggcc 360 cccgtgcccg cag gtg aga ccg tgg agc cgc ccc cgc cgg cgc agc tgc 409 Val Arg Pro Trp Ser Arg Pro Arg Arg Arg Ser Cys 1 5 10 act tca tgt acg tg 423 Thr Ser Cys Thr 15 13 3494 DNA H. sapiens 13 cacgctctga tgccgccaag cgcctgctgg aggccagcgc agatgccaac atccaggaca 60 acatgggccg caccccgctg catgcggctg tgtctgccga cgcacaaggt gtcttccaga 120 tcctgatccg gaaccgagcc acagacctgg atgcccgcat gcatgatggc acgacgccac 180 tgatcctggc tgcccgcctg gccgtggagg gcatgctgga ggacctcatc aactcacacg 240 ccgacgtcaa cgccgtagat gacctgggca agtccgccct gcactgggcc gccgccgtga 300 acaatgtgga tgccgcagtt gtgctcctga agaacggggc taacaaagat atgcagaaca 360 acagggagga gacacccctg tttctggccg cccgggaggg cagctacgag accgccaagg 420 tgctgctgga ccactttgcc aaccgggaca tcacggatca tatggaccgc ctgccgcgcg 480 acatcgcaca ggagcgcatg catcacgaca tcgtgaggct gctggacgag tacaacctgg 540 tgcgcagccc gcagctgcac ggagccccgc tggggggcac gcccaccctg tcgcccccgc 600 tctgctcgcc caacggctac ctgggcagcc tcaagcccgg cgtgcagggc aagaaggtcc 660 gcaagcccag cagcaaaggc ctggcctgtg gaagcaagga ggccaaggac ctcaaggcac 720 ggaggaagaa gtcccaggat ggcaagggct gcctgctgga cagctccggc atgctctcgc 780 ccgtggactc cctggagtca ccccatggct acctgtcaga cgtggcctcg ccgccactgc 840 tgccctcccc gttccagcag tctccgtccg tgcccctcaa ccacctgcct gggatgcccg 900 acacccacct gggcatcggg cacctgaacg tggcggccaa gcccgagatg gcggcgctgg 960 gtgggggcgg ccggctggcc tttgagactg gcccacctcg tctctcccac ctgcctgtgg 1020 cctctggcac cagcaccgtc ctgggctcca gcagcggagg ggccctgaat ttcactgtgg 1080 gcgggtccac cagtttgaat ggtcaatgcg agtggctgtc ccggctgcag agcggcatgg 1140 tgccgaacca atacaaccct ctgcggggga gtgtggcacc aggccccctg agcacacagg 1200 ccccctccct gcagcatggc atggtaggcc cgctgcacag tagccttgct gccagcgccc 1260 tgtcccagat gatgagctac cagggcctgc ccagcacccg gctggccacc cagcctcacc 1320 tggtgcagac ccagcaggtg cagccacaaa acttacagat gcagcagcag aacctgcagc 1380 cagcaaacat ccagcagcag caaagcctgc agccgccacc accaccacca cagccgcacc 1440 ttggcgtgag ctcagcagcc agcggccacc tgggccggag cttcctgagt ggagagccga 1500 gccaggcaga cgtgcagcca ctgggcccca gcagcctggc ggtgcacact attctgcccc 1560 aggagagccc cgccctgccc acgtcgctgc catcctcgct ggtcccaccc gtgaccgcag 1620 cccagttcct gacgcccccc tcgcagcaca gctactcctc gcctgtggac aacaccccca 1680 gccaccagct acaggtgcct gagcacccct tcctcacccc gtcccctgag tcccctgacc 1740 agtggtccag ctcgtccccg cattccaacg tctccgactg gtccgagggc gtctccagcc 1800 ctcccaccag catgcagtcc cagatcgccc gcattccgga ggccttcaag taaacggcgc 1860 gccccacgag accccggctt cctttcccaa gccttcgggc gtctgtgtgc gctctgtgga 1920 tgccagggcc gaccagagga gcctttttaa aacacatgtt tttatacaaa ataagaacaa 1980 ggattttaat tttttttagt atttatttat gtacttttat tttacacaga aacactgcct 2040 ttttatttat atgtactgtt ttatctggcc ccaggtagaa acttttatct attctgagaa 2100 aacaagcaag ttctgagagc cagggttttc ctacgtagga tgaaaagatt cttctgtgtt 2160 tataaaatat aaacaaagat tcatgattta taaatgccat ttatttattg attccttttt 2220 tcaaaatcca aaaagaaatg atgttggaga agggaagttg aacgagcata gtccaaaaag 2280 ctcctggggc gtccaggccg cgccctttcc ccgacgccca cccaacccca agccagcccg 2340 gccgctccac cagcatcacc tgcctgttag gagaagctgc atccagaggc aaacggaggc 2400 aaagctggct caccttccgc acgcggatta atttgcatct gaaataggaa acaagtgaaa 2460 gcatatgggt tagatgttgc catgtgtttt agatggtttc ttgccagcat gcttgtgaaa 2520 atgtgttctc ggagtgtgta tgccaagagt gcacccatgg taccaatcat gaatctttgt 2580 ttcaggttca gtattatgta gttgttcgtt ggttatacaa gttcttggtc cctccagaac 2640 caccccggcc ccctgcccgt tcttgaaatg taggcatcat gcatgtcaaa catgagatgt 2700 gtggactgtg gcacttgcct gggtcacaca cggaggcatc ctaccctttt ctggggaaag 2760 acactgcctg ggctgacccc ggtggcggcc ccagcacctc agcctgcaca gtgtccccca 2820 ggttccgaag aagatgctcc agcaacacag cctgggcccc agctcgcggg acccgacccc 2880 ccgtgggctc ccgtgttttg taggagactt gccagagccg ggcacattga gctgtgcaac 2940 gccgtgggct gcgtcctttg gtcctgtccc cgcagccctg gcagggggca tgcggtcggg 3000 caggggctgg agggaggcgg gggctgccct tgggccaccc ctcctagttt gggaggagca 3060 gatttttgca ataccaagta tagcctatgg cagaaaaaat gtctgtaaat atgtttttaa 3120 aggtggattt tgtttaaaaa atcttaatga atgagtctgt tgtgtgtcat gccagtgagg 3180 gacgtcagac ttggctcagc tcggggagcc ttagccgccc atgcactggg gacgctccgc 3240 tgccgtgccg cctgcactcc tcagggcagc ctcccccggc tctacggggg ccgcgtggtg 3300 ccatccccag ggggcatgac cagatgcgtc ccaagatgtt gatttttact gtgttttata 3360 aaatagagtg tagtttacag aaaaagactt taaaagtgat ctacatgagg aactgtagat 3420 gatgtatttt tttcatcttt tttgttaact gatttgcaat aaaaatgata ctgatggtga 3480 aaaaaaaaaa aaaa 3494 14 20 DNA Artificial Sequence Antisense Oligonucleotide 14 cgtgcgtccc tcttagggtc 20 15 20 DNA Artificial Sequence Antisense Oligonucleotide 15 cacagcagac ctgggcaggc 20 16 20 DNA Artificial Sequence Antisense Oligonucleotide 16 cagccctccc ctaatgagac 20 17 20 DNA Artificial Sequence Antisense Oligonucleotide 17 cggccacgca ctgtgcaggc 20 18 20 DNA Artificial Sequence Antisense Oligonucleotide 18 acggtctcac ctgcgggcac 20 19 20 DNA Artificial Sequence Antisense Oligonucleotide 19 tcacttgagg cccacggagt 20 20 20 DNA Artificial Sequence Antisense Oligonucleotide 20 cactgcctac ctggaagaca 20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21 accacctgcg tcaccacatt 20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22 cacgatttcc ctgaccagcc 20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23 aagggcaggc actggccacc 20 24 20 DNA Artificial Sequence Antisense Oligonucleotide 24 ttgcagttgt ttcctggaca 20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 ttctggcagg catttggcat 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26 tggcacagca gacctgtgcg 20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 tgaggtggca cagcagacct 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28 tccacgtcct ggctgcaggc 20 29 20 DNA Artificial Sequence Antisense Oligonucleotide 29 tccccaatct ggtccaggca 20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 ggaactcccc aatctggtcc 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31 ccatcgatct tgtccagaca 20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 gttgttgttg atgtcacagt 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33 cactcgttgt tgttgatgtc 20 34 20 DNA Artificial Sequence Antisense Oligonucleotide 34 ggcaggcagt cgcagaaggc 20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 aagccgggca ggcagtcgca 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36 gtgtagctgt ccacgcagtc 20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 gcaggtgcac gtgtagctgt 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38 gcacaaggtt ctggcagttg 20 39 20 DNA Artificial Sequence Antisense Oligonucleotide 39 gcagccacct cacaggacac 20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 gccctccatg ctggcacagg 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41 acagagccct ccatgctggc 20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 gggcaggagc acttgtaggt 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43 ggcactcgca gtggaagtca 20 44 20 DNA Artificial Sequence Antisense Oligonucleotide 44 cctcgaagcc cgcagggcac 20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 tcggatgtgg gctcacaggt 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46 gtagtccagg atgtggcaca 20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 aagctgtagt ccaggatgtg 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48 ttgaagttga gggagcagtc 20 49 20 DNA Artificial Sequence Antisense Oligonucleotide 49 ggtcattgaa gttgagggag 20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 tgcgtgcagt tcttccaggg 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51 gagactgcgt gcagttcttc 20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 tggctgtcac agtggccgtc 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53 tgcactggct gtcacagtgg 20 54 20 DNA Artificial Sequence Antisense Oligonucleotide 54 tggtccttgc agtactggtc 20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 tgaagtggtc cttgcagtac 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 ccacgttggt gtgcagcacg 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 gaagaccacg ttggtgtgca 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58 cgcttgaaga ccacgttggt 20 59 20 DNA Artificial Sequence Antisense Oligonucleotide 59 gggaagatca tctgctggcc 20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 ctctggaagc actgcgagga 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 tggcactctg gaagcactgc 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 ttgcgggaca gcagcacccc 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63 aaccagagct ggccatgctg 20 64 20 DNA Artificial Sequence Antisense Oligonucleotide 64 cagggaacca gagctggcca 20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 aacttcttgg tctccaggtc 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 accggaactt cttggtctcc 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 gcagacatgc gcaggtcagc 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68 ccatggcaga catgcgcagg 20 69 20 DNA Artificial Sequence Antisense Oligonucleotide 69 gcggtctgtc tggttgtgca 20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 ttggcatctg cgctggcctc 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 tgatgaggtc ctccagcatg 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 tgagttgatg aggtcctcca 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73 gcggacttgc ccaggtcatc 20 74 20 DNA Artificial Sequence Antisense Oligonucleotide 74 ccgttcttca ggagcacaac 20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 atccgtgatg tcccggttgg 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 tagccgttgg gcgagcagag 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 ctccgtgcct tgaggtcctt 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78 tcttcctccg tgccttgagg 20 79 20 DNA Artificial Sequence Antisense Oligonucleotide 79 cagcccttgc catcctggga 20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 gtctgcacca ggtgaggctg 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 tgctgggtct gcaccaggtg 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 gcacctgctg ggtctgcacc 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83 tggctgcacc tgctgggtct 20 84 20 DNA Artificial Sequence Antisense Oligonucleotide 84 tcgagctatt acttgaacgc 20 85 20 DNA Artificial Sequence Antisense Oligonucleotide 85 gctgctggca cctcgagcta 20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86 ttcacatgac accgatcaat 20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87 ctttaaggac ggagggatag 20 88 20 DNA Artificial Sequence Antisense Oligonucleotide 88 tctgtgtaaa ataaaagtac 20 89 20 DNA Artificial Sequence Antisense Oligonucleotide 89 tgctcgttca acttcccttc 20 90 20 DNA Artificial Sequence Antisense Oligonucleotide 90 ctggagcatc ttcttcggaa 20 91 20 DNA Artificial Sequence Antisense Oligonucleotide 91 cccgagctga gccaagtctg 20 92 20 DNA H. sapiens 92 gcctgcacag tgcgtggccg 20 93 20 DNA H. sapiens 93 actccgtggg cctcaagtga 20 94 20 DNA H. sapiens 94 aatgtggtga cgcaggtggt 20 95 20 DNA H. sapiens 95 ggtggccagt gcctgccctt 20 96 20 DNA H. sapiens 96 tgtccaggaa acaactgcaa 20 97 20 DNA H. sapiens 97 atgccaaatg cctgccagaa 20 98 20 DNA H. sapiens 98 aggtctgctg tgccacctca 20 99 20 DNA H. sapiens 99 gcctgcagcc aggacgtgga 20 100 20 DNA H. sapiens 100 tgcctggacc agattgggga 20 101 20 DNA H. sapiens 101 tgtctggaca agatcgatgg 20 102 20 DNA H. sapiens 102 actgtgacat caacaacaac 20 103 20 DNA H. sapiens 103 gacatcaaca acaacgagtg 20 104 20 DNA H. sapiens 104 gccttctgcg actgcctgcc 20 105 20 DNA H. sapiens 105 tgcgactgcc tgcccggctt 20 106 20 DNA H. sapiens 106 caactgccag aaccttgtgc 20 107 20 DNA H. sapiens 107 cctgtgccag catggagggc 20 108 20 DNA H. sapiens 108 gccagcatgg agggctctgt 20 109 20 DNA H. sapiens 109 tgacttccac tgcgagtgcc 20 110 20 DNA H. sapiens 110 acctgtgagc ccacatccga 20 111 20 DNA H. sapiens 111 tgtgccacat cctggactac 20 112 20 DNA H. sapiens 112 cacatcctgg actacagctt 20 113 20 DNA H. sapiens 113 gacggccact gtgacagcca 20 114 20 DNA H. sapiens 114 ccactgtgac agccagtgca 20 115 20 DNA H. sapiens 115 gaccagtact gcaaggacca 20 116 20 DNA H. sapiens 116 cgtgctgcac accaacgtgg 20 117 20 DNA H. sapiens 117 tgcacaccaa cgtggtcttc 20 118 20 DNA H. sapiens 118 accaacgtgg tcttcaagcg 20 119 20 DNA H. sapiens 119 tcctcgcagt gcttccagag 20 120 20 DNA H. sapiens 120 gcagtgcttc cagagtgcca 20 121 20 DNA H. sapiens 121 ggggtgctgc tgtcccgcaa 20 122 20 DNA H. sapiens 122 cagcatggcc agctctggtt 20 123 20 DNA H. sapiens 123 gacctggaga ccaagaagtt 20 124 20 DNA H. sapiens 124 ggagaccaag aagttccggt 20 125 20 DNA H. sapiens 125 gctgacctgc gcatgtctgc 20 126 20 DNA H. sapiens 126 cctgcgcatg tctgccatgg 20 127 20 DNA H. sapiens 127 gaggccagcg cagatgccaa 20 128 20 DNA H. sapiens 128 catgctggag gacctcatca 20 129 20 DNA H. sapiens 129 tggaggacct catcaactca 20 130 20 DNA H. sapiens 130 gatgacctgg gcaagtccgc 20 131 20 DNA H. sapiens 131 gttgtgctcc tgaagaacgg 20 132 20 DNA H. sapiens 132 ccaaccggga catcacggat 20 133 20 DNA H. sapiens 133 ctctgctcgc ccaacggcta 20 134 20 DNA H. sapiens 134 aaggacctca aggcacggag 20 135 20 DNA H. sapiens 135 cctcaaggca cggaggaaga 20 136 20 DNA H. sapiens 136 tcccaggatg gcaagggctg 20 137 20 DNA H. sapiens 137 cagcctcacc tggtgcagac 20 138 20 DNA H. sapiens 138 cacctggtgc agacccagca 20 139 20 DNA H. sapiens 139 agacccagca ggtgcagcca 20 140 20 DNA H. sapiens 140 tagctcgagg tgccagcagc 20 141 20 DNA H. sapiens 141 attgatcggt gtcatgtgaa 20 142 20 DNA H. sapiens 142 gtacttttat tttacacaga 20 143 20 DNA H. sapiens 143 gaagggaagt tgaacgagca 20 144 20 DNA H. sapiens 144 ttccgaagaa gatgctccag 20 145 20 DNA H. sapiens 145 cagacttggc tcagctcggg 20 146 20 DNA Artificial Sequence Antisense Oligonucleotide 146 nnnnnnnnnn nnnnnnnnnn 20 

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targeted to a nucleic acid molecule encoding Notch1, wherein said compound specifically hybridizes with said nucleic acid molecule encoding Notch1 and inhibits the expression of Notch1.
 2. The compound of claim 1 which is an antisense oligonucleotide.
 3. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified internucleoside linkage.
 4. The compound of claim 3 wherein the modified internucleoside linkage is a phosphorothioate linkage.
 5. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified sugar moiety.
 6. The compound of claim 5 wherein the modified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
 7. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified nucleobase.
 8. The compound of claim 7 wherein the modified nucleobase is a 5-methylcytosine.
 9. The compound of claim 2 wherein the antisense oligonucleotide is a chimeric oligonucleotide.
 10. A compound 8 to 80 nucleobases in length which specifically hybridizes with at least an 8-nucleobase portion of a preferred target region on a nucleic acid molecule encoding Notch1.
 11. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier or diluent.
 12. The composition of claim 11 further comprising a colloidal dispersion system.
 13. The composition of claim 11 wherein the compound is an antisense oligonucleotide.
 14. A method of inhibiting the expression of Notch1 in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of Notch1 is inhibited.
 15. A method of treating an animal having a disease or condition associated with Notch1 comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of Notch1 is inhibited.
 16. The method of claim 15 wherein the disease or condition is a developmental disorder.
 17. The method of claim 15 wherein the disease or condition is an autoimmune disorder.
 18. The method of claim 15 wherein the disease or condition arises from aberrant apoptosis.
 19. The method of claim 15 wherein the disease or condition is a hyperproliferative disorder.
 20. The method of claim 19 wherein the hyperproliferative disorder is cancer.
 21. A method for inducing apoptosis in a cell or animal comprising administering to a cell or animal the compound of claim
 1. 22. A method for treating a subject having a disease or condition associated with insufficient apoptosis comprising administering the compound of claim 1 in an amount effective to reduce Notch1 levels or activity.
 23. The method of claim 22 wherein the condition associated with insufficient apoptosis is a hyperproliferative condition.
 24. A pharmaceutical composition comprising the compound of claim 1 and another active ingredient for inducing apoptosis.
 25. A kit comprising the compound of claim 1 and instructions for said compound in the induction of apoptosis.
 26. A method for increasing caspase activity in a cell or animal comprising administering to a cell or animal the compound of claim
 1. 